Devices For Wound Healing

ABSTRACT

The present invention provides devices and methods for improving wound healing, in particular, in diabetic subjects.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61/991,201, filed May 9, 2014, which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

The invention was supported, in whole, or in part, by NIH grant numbers 1 R24 DK091210-01A1, RO1 DE017413-01A1, and RO1 DK98055-06A1. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to hydrogels for cell therapy and their use in wound healing.

BACKGROUND OF THE INVENTION

Diabetes is a chronic disease in which the body cannot properly regulate glucose metabolism. An estimated 9% of the U.S. population has been diagnosed with diabetes. Because of complications associated with diabetes, such as neuropathy, a weakened immune system, and narrowed arteries, wounds in diabetic subjects are particularly difficult to heal—they are prone to infection and either do not heal or heal slowly. Thus, there is a need for compositions that are effective in healing wounds such as those from diabetic subjects.

Cell transplantation has been used in regenerative medicine for musculoskeletal disorders as well as degenerative conditions such as diabetes with limited success. Limitations of earlier approaches include loss of cell viability and function following transplantation.

Currently available products for the management of diabetic foot ulceration include the growth factor, becaplermin (REGRANEX™), and bioengineered skins, APLIGRAF™ and DERMAGRAFT™. However, efficiency of these products to heal ulcers has been limited. Thus, there is a need for a more efficient therapy that is also easier and less expensive to produce. This invention addresses these needs.

SUMMARY OF THE INVENTION

The invention overcomes the drawbacks of earlier products described above and features a device comprising a structural composition (e.g., a hydrogel scaffold or cell delivery vehicle) and a population of fibroblasts, where the hydrogel comprises pores, and where the population of fibroblasts is seeded into or onto the hydrogel, e.g., an alginate hydrogel. Preferably, the fibroblasts are derived from or isolated from a subject diagnosed with or suffering from diabetes. For example, the fibroblasts are derived from or isolated from an ulcer, e.g., a foot ulcer, on a subject diagnosed with or suffering from diabetes. As described herein, hydrogels comprising diabetic ulcer fibroblast cells from diabetic wounds, e.g., foot ulcers, promote diabetic wound healing better than nondiabetic, nonulcerated foot-derived fibroblasts.

The device contains nanopores, micropores, macropores, or a combination thereof. The size of the pores permits cell migration or movement (e.g., fibroblast migration into and/or egress out of the delivery vehicle) through the pores. For example, the composition comprises pores that are characterized by a diameter of 20-500 μm (e.g., 50-500 μm, or 20-300 μm).

The population of fibroblasts comprises a fibroblast that is derived from or isolated from a subject diagnosed as suffering from diabetes. In some embodiments, the population of fibroblasts comprises a fibroblast that is derived from or isolated from a subject having a wound, e.g., at or near the site of a skin ulcer. For example, a site near a skin ulcer is about 0.1 mm, 0.5 mm, 1 mm, 2.5 mm, 5 mm, 10 mm, 15 mm, 20 mm, or 40 mm away from a perimeter or margin of the ulcer. For example, the wound is located in an extremity (e.g., an arm, hand, leg, foot, toe, or finger), and the cells are explanted or obtained directly from the ulcerated skin or lesion itself or about 0.1 mm, 0.5 mm, 1 mm, 2.5 mm, 5 mm, 10 mm, 15 mm, 20 mm, or 40 mm from a perimeter or margin of the ulcer. In some cases, the wound is a diabetic wound. Optionally, the diabetic wound is characterized by inflammation (e.g., presence of pro-inflammatory immune cells and pro-inflammatory cytokines). Exemplary pro-inflammatory immune cells include macrophages, dendritic cells, T cells (helper T cells, CD8+cytotoxic T cells), and natural killer cells. Exemplary pro-inflammatory cytokines include tumor necrosis factor-a (TNFα), IL-1, IL-2, and interferon-γ (IFN-γ). In some cases, the wound is an ulcer (e.g., a foot ulcer).

The subject in need of a device of the invention has been diagnosed with diabetes and suffers from a wound, e.g., a dermal wound.

In some cases, the fibroblast of the device is derived from or isolated from the site of the wound or a site adjacent to the wound. In some examples, fibroblasts derived from or isolated from diabetic wounds have an altered production (e.g., expression level and/or expression pattern) of proteins that are important for making a well-structured wound bed. In some cases, fibroblasts derived from diabetic wounds have an absence of stimulatory factors that are linked to the non-healing features of these wounds.

At least 5% (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more) of the fibroblasts in the population are derived from or isolated from the site of a wound or a site adjacent to the wound. For example, the population of fibroblasts comprises a fibroblast that is derived from a portion of the skin of the subject. In some cases, the population of fibroblasts is derived from or isolated from a foot ulcer from a subject suffering from diabetes.

A diabetic ulcer fibroblast is identified by differential expression of biomarkers or differential cell signaling responses compared to normal fibroblasts or fibroblasts taken or derived from non-ulcerous tissue. For example, the population of fibroblasts derived from diabetic foot ulcers expresses fibronectin at a level at least 1.1 fold more than nondiabetic, nonulcerated foot-derived fibroblasts, e.g., at least 1.2 fold more, at least 1.3 fold more, at least 1.4 fold more, at least 1.5 fold more, at least 1.6 fold more, at least 1.7 fold more, at least 1.8 fold more, at least 1.9 fold more, at least 2 fold more, at least 3 fold more, at least 4 fold more, at least 5 fold more, at least 6 fold more, at least 7 fold more, at least 8 fold more, at least 9 fold more, at least 10 fold more, at least 11 fold more, at least 12 fold more, at least 13 fold more, at least 14 fold more, at least 15 fold more, at least 20 fold more, at least 30 fold more, at least 40 fold more, at least 50 fold more, at least 60 fold more, at least 70 fold more, at least 80 fold more, at least 90 fold more, or at least 100 fold more.

For example, the subject is a mammal, e.g., a human, dog, cat, pig, or horse. Preferably, the subject is a human.

In some cases, the population of fibroblasts comprises fibroblasts that have been cultured and optionally expanded in vitro.

In some embodiments, the population of fibroblasts includes a fibroblast comprising metabolic memory. For example, the metabolic memory is associated with an epigenetic alteration or is due to an epigenetic alteration compared to a fibroblast derived from a subject i) not suffering from diabetes, ii) not having a wound, or both i) and ii).

In some embodiments, the population of fibroblasts includes a fibroblast comprising an epigenetic alteration compared to a fibroblast derived from a subject i) not suffering from diabetes, ii) not having a wound, or both i) and ii). In some examples, fibroblasts of diabetic patients, in particular, in fibroblasts from foot ulcers of diabetic patients, comprise epigenetic changes, e.g., methylation changes.

Optionally, the population of fibroblasts comprises a genetically modified fibroblast. For example, the fibroblasts are modified to overexpress growth factors or cytokines that enhance wound healing (e.g., angiogenic factors such as vascular endothelial growth factor (VEGF), placental growth factor (PlGF), fibroblast growth factor (FGF)), increase epithelial or fibroblast migration (e.g., hepatocyte growth factor (HGF)), modify the inflammatory/immune response (e.g., transforming growth factor beta (TGF-β), interleukin-10 (IL-10)), or inhibit scarring.

Some devices of the invention further comprise a bioactive composition. Exemplary bioactive compositions include cell growth and/or cell differentiation factors. For example, a bioactive composition includes a growth factor, morphogen, differentiation factor, and/or chemoattractant. For example, the device includes vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), or fibroblast growth factor 2 (FGF2) or a combination thereof. Other bioactive compositions include hormones, neurotransmitters, neurotransmitter or growth factor receptors, interferons, interleukins, chemokines, MMP-sensitive substrate, cytokines, colony stimulating factors and phosphatase inhibitors. Growth factors used to promote angiogenesis, wound healing, and/or tissue regeneration can be included in the device.

In some embodiments, the device is implantable or injectable into a subject.

In addition, the invention features a method of treating a wound in a patient in need thereof comprising administering a device described herein. For example, the method includes the step of providing a diabetic ulcer fibroblast, seeding a hydrogel such as an alginate hydrogel with the fibroblast and administering the cell-seeded hydrogel to a diabetic skin wound. For example, the cell-seeded hydrogel is administered to or near the wound, e.g., skin ulcer. A site near a skin ulcer is about 0.1 mm, 0.5 mm, 1 mm, 2.5 mm, 5 mm, 10 mm, 15 mm, 20 mm, or 40 mm away from a perimeter or margin of the ulcer. For example, the wound is located in an extremity (e.g., an arm, hand, leg, foot, toe, or finger), and the cell seeded hydrogel is administered directly to the ulcerated skin or lesion itself or about 0.1 mm, 0.5 mm, 1 mm, 2.5 mm, 5 mm, 10 mm, 15 mm, 20 mm, or 40 mm from a perimeter or margin of the ulcer.

For example, the patient suffers from diabetes. For example, the patient suffers from a wound that is resistant to healing. In some cases, the wound is located in an extremity of the patient (e.g., an arm, leg, foot, hand, toe, or finger). For example, the patient suffers from an ulcer, e.g., in an extremity such as a foot. Exemplary ulcers have a diameter of at least about 25 mm, 50 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, or greater.

Routes of administration of the device include injection or implantation. Alternate routes include topical application, e.g., applying the device in the form of a coating, covering, or bandage contacting a wound. Other routes of administration comprise spraying the device, e.g., hydrogel, onto a wound, e.g., as a fluid or aerosol, followed by solidification of the device, e.g., hydrogel, once in contact with the wound.

The dermal diabetic ulcer-associated fibroblasts are purified, e.g., by separating the fibroblasts from other cellular or non-cellular material. The fibroblasts can be purified or in a heterologous mixture of cells taken from or adjacent to a diabetic wound. In some embodiments, the population of fibroblasts comprises an autologous fibroblast (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more autologous fibroblasts). Alternatively or in addition, the population of fibroblasts comprises an allogeneic or xenogeneic fibroblast. For example, the population of fibroblasts comprises at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more) allogeneic fibroblasts. For example, the population of fibroblasts comprises at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more) xenogeneic fibroblasts.

The fibroblasts preferably elicit a minimal adverse host response (e.g., minimal harmful inflammation and/or minimal host immune rejection of the transplanted fibroblasts).

The devices of the invention enhance the viability of passenger cells (e.g., fibroblasts) and induce their outward migration to populate injured or defective bodily tissues enhance the success of tissue regeneration, e.g., the regeneration of muscle tissue or other tissues, as well as angiogenesis. Such a device that controls cell function and/or behavior, e.g., locomotion, contains a scaffold composition and one or more bioactive compositions. The bioactive composition is incorporated into or coated onto the scaffold composition. The scaffold composition and/or bioactive composition temporally and spatially (directionally) controls egress of a resident cell (e.g., fibroblast) or progeny thereof. At the end of a treatment period, the device is has release a substantial number of the passenger cells that were originally used to seed the device, e.g., there is a net efflux of passenger cells. For example, the device releases 10% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or more) of the seeded passenger cells by the end of a treatment period compared to at the commencement of treatment. In another example, the device contains 50% or less (e.g., 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2.5%, 1%, or less) of the seeded passenger cells at the end of a treatment period compared to at the commencement of treatment. In some cases, a greater number of cells can be released than originally loaded if the cells proliferate after being placed in contact with the scaffold.

In some cases, the devices mediate modification and release of host cells from the material in vivo, thereby improving the function of cells that have resided in the scaffold composition. For example, the scaffold composition temporally and spatially (directionally) controls fibroblast migration. For example, the scaffold composition mediates release of fibroblasts from the material in vivo.

This device includes a scaffold composition which incorporates or is coated with a bioactive composition; the device regulates the egress of resident cells. Egress is regulated spatially and temporally. Depending on the application for which the device is designed, the device regulates egress through the physical or chemical characteristics of the scaffold composition itself. For example, the scaffold composition is differentially permeable, allowing cell egress only in certain physical areas of the scaffold composition. The permeability of the scaffold composition is regulated, for example, by selecting or engineering a material for greater or smaller pore size, density, polymer cross-linking, stiffness, toughness, ductility, or viscoelascticity. The scaffold composition contains physical channels or paths through which cells can move more easily towards a targeted area of egress of the device or of a compartment within the device. The scaffold composition is optionally organized into compartments or layers, each with a different permeability, so that the time required for a cell to move through the device is precisely and predictably controlled. Migration is also regulated by the degradation, de- or re-hydration, oxygenation, chemical or pH alteration, or ongoing self-assembly of the scaffold composition. These processes are driven by diffusion or cell-secretion of enzymes or other reactive chemicals.

Alternatively or in addition, egress is regulated by a bioactive composition. By varying the concentration of growth factors, homing/migration factors, morphogens, differentiation factors, oligonucleotides, hormones, neurotransmitters, neurotransmitter or growth factor receptors, interferons, interleukins, chemokines, cytokines, colony stimulating factors, chemotactic factors, extracellular matrix components, adhesion molecules and other bioactive compounds in different areas of the device. The device controls and directs the migration of cells through its structure. Chemical affinities are used to channel cells towards a specific area of egress. For example, adhesion molecules are used to attract or retard the migration of cells. By varying the density and mixture of those bioactive substances, the device controls the timing of the migration and egress. The density and mixture of these bioactive substances is controlled by initial doping levels or concentration gradient of the substance, by embedding the bioactive substances in scaffold material with a known leaching rate, by release as the scaffold material degrades, by diffusion from an area of concentration, by interaction of precursor chemicals diffusing into an area, or by production/excretion of compositions by resident support cells. The physical or chemical structure of the scaffold composition also regulates the diffusion of bioactive agents through the device.

The bioactive composition includes one or more compounds that regulate cell function and/or behavior. For example, the bioactive composition includes cell adhesion ligands (e.g., RGD-containing peptides) and growth factors (e.g., FGF and HGF). The bioactive composition is covalently linked to the scaffold composition or non-covalently associated with the scaffold. For example, the bioactive composition is an extracellular matrix (ECM) component that is chemically crosslinked to the scaffold composition. Regardless of the tissue of origin, ECM components generally include three general classes of macromolecules: collagens, proteoglycans/glycosaminoglycans (PG/GAG), and glycoproteins, e.g., fibronectin (FN), laminin, and thrombospondin. ECM components associate with molecules on the cell surface and mediate adhesion and/or motility. Preferably, the ECM component associated with the scaffold composition is a proteoglycan attachment peptide or cyclic peptide containing the amino acid sequence arginine-glycine-aspartic acid (RGD). Proteoglycan attachment peptides are selected from the group consisting of G₄RGDSP (SEQ ID NO: 1), XBBXBX (SEQ ID NO: 2), PRRARV (SEQ ID NO: 3), YEKPGSPPREVVPRPRPGV (SEQ ID NO:4), RPSLAKKQRFRHRNRKGYRSQRGHSRGR (SEQ ID NO: 5), and RIQNLLKITNLRIKFVK (SEQ ID NO: 6), and cell attachment peptides are selected from the group consisting of RGD, RGDS (SEQ ID NO: 7), LDV, REDV (SEQ ID NO: 8), RGDV (SEQ ID NO: 9), LRGDN (SEQ ID NO: 10), IKVAV (SEQ ID NO: 11), YIGSR (SEQ ID NO: 12), PDSGR (SEQ ID NO: 13), RNIAEIIKDA (SEQ ID NO: 14), RGDT (SEQ ID NO: 15), DGEA (SEQ ID NO: 16), and VTXG (SEQ ID NO: 17).

Components of the ECM, e.g., FN, laminin, and collagen, interact with the cell surface via the integrin family of receptors, a group of divalent cation-dependent cell surface glycoproteins that mediate cellular recognition and adhesion to components of the ECM and to other cells. Ligands recognized by integrins typically contain an RGD amino acid sequence that is expressed in many ECM proteins. Exemplary molecules that mediate cell adhesion and/or movement include FN, laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix protein, fibronogen, fibrin, fibulin, mucins, entactin, osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin, versican, von Willebrand Factor, polysacchride heparin sulfate, cell adhesion molecules including connexins, selectinsinclude collagen, RGD (Arg-Gly-Asp) and YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides, glycosaminoglycans (GAGs), hyaluronic acid (HA), integrins, selectins, cadherins and members of the immunoglobulin superfamily. Carbohydrate ligands of the ECM include the polysaccharides hyaluronic acid, and chondroitin-6-sulfate.

Signal transduction events that participate in the process of cell motility are initiated in response to cell growth and/or cell differentiation factors. Thus, the device optionally contains a second bioactive composition that is a growth factor, morphogen, differentiation factor, or chemoattractant. For example, the device includes vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), or fibroblast growth factor 2 (FGF2) or a combination thereof. Other factors include hormones, neurotransmitters, neurotransmitter or growth factor receptors, interferons, interleukins, chemokines, MMP-sensitive substrate, cytokines, colony stimulating factors. Growth factors used to promote angiogenesis, bone regeneration, wound healing, and other aspects of tissue regeneration are listed herein and are used alone or in combination to induce colonization or regeneration of bodily tissues by cells that have migrated out of an implanted device.

The scaffold composition is biocompatible. The composition is bio-degradable/erodable or resistant to breakdown in the body. Relatively permanent (degradation resistant) scaffold compositions include metals and some polymers such as silk. Preferably, the scaffold composition degrades at a predetermined rate based on a physical parameter selected from the group consisting of temperature, pH, hydration status, and porosity, the cross-link density, type, and chemistry or the susceptibility of main chain linkages to degradation or it degrades at a predetermined rate based on a ratio of chemical polymers. For example, a high molecular weight polymer comprised of solely lactide degrades over a period of years, e.g., 1-2 years, while a low molecular weight polymer comprised of a 50:50 mixture of lactide and glycolide degrades in a matter of weeks, e.g., 1, 2, 3, 4, 6, 10 weeks. A calcium cross-linked gels composed of high molecular weight, high guluronic acid alginate degrade over several months (1, 2, 4, 6, 8, 10, 12 months) to years (1, 2, 5 years) in vivo, while a gel comprised of low molecular weight alginate, and/or alginate that has been partially oxidized, will degrade in a matter of weeks.

In one example, cells mediate degradation of the scaffold matrix, i.e., the scaffold composition is enzymatically digested by a composition elicited by a resident cell, and the egress of the cell is dependent upon the rate of enzymatic digestion of the scaffold composition. In this case, polymer main chains or cross-links contain compositions, e.g., oligopeptides, that are substrates for collagenase or plasmin, or other enzymes produced by within or adjacent to the scaffold composition.

Exemplary scaffold compositions include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, polyanhydrides, polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers, pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers or graft copolymers of any of the above. One preferred scaffold composition includes an RGD-modified alginate.

Porosity of the scaffold composition influences migration of the cells through the device and egress of the cells from the device. Pores are nanoporous, microporous, or macroporous. In some cases, the pores are a combination of these sizes. For example, the pores of the scaffold composition are large enough for a cell, e.g., fibroblast, to migrate through. For example, the diameter of nanopores are less than about 10 nm; micropore are in the range of about 100 nm-20 μm in diameter; and, macropores are greater than about 20 μm (preferably greater than about 100 μm and even more preferably greater than about 400 μm). In one example, the scaffold composition is macroporous with aligned pores of about 400-500 μm in diameter.

The devices are manufactured in their entirety in the absence of cells or can be assembled around or in contact with cells (the material is gelled or assembled around cells in vitro or in vivo in the presence of cells and tissues) and then contacted with cells to produce a cell-seeded structure. Alternatively, the device is manufactured in two or more (3, 4, 5, 6, . . . 10 or more) stages in which one layer or compartment is made and seeded with cells followed by the construction of a second, third, fourth or more layers, which are in turn seeded with cells in sequence. Each layer or compartment is identical to the others or distinguished from one another by the number, genotype, or phenotype of the seed cell population as well as distinct chemical, physical and biological properties. Prior to implantation, the device is contacted with purified populations cells or characterized mixtures of cells as described above. Preferably, the cells are human; however, the system is adaptable to other eukaryotic animal cells, e.g., canine, feline, equine, bovine, and porcine as well as prokaryotic cells such as bacterial cells.

A method of making a device is carried out by providing a scaffold composition and covalently linking or noncovalently associating the scaffold composition with a first bioactive composition. The first bioactive composition preferably contains a cell adhesion ligand. The scaffold composition is also contacted with a second bioactive composition. The second bioactive composition is preferably non-covalently associated with the scaffold composition to yield a doped scaffold, i.e., a scaffold composition that includes one or more bioactive substances. The contacting steps are optionally repeated to yield a plurality of doped scaffolds, e.g., each of the contacting steps is characterized by a different amount of the second bioactive composition to yield a gradient of the second bioactive composition in the device. Rather than altering the amount of composition, subsequent contacting steps involve a different bioactive composition, i.e., a third, fourth, fifth, sixth . . . , composition or mixture of compositions, that is distinguished from the prior compositions or mixtures of prior doping steps by the structure or chemical formula of the factor(s). The method optionally involves adhering individual niches, layers, or components to one another and/or insertion of semi-permeable, permeable, or nonpermeable membranes within or at one or more boundaries of the device to further control/regulate locomotion of cells or bioactive compositions. As described above, the device is seeded with cells after completion of the construction of the device or in an iterative manner throughout the construction of each component.

Therapeutic applications of the device include tissue generation, regeneration/repair, as well as augmentation of function of a mammalian bodily tissue in and around a wound. For example, the method includes the steps of providing a device that includes scaffold composition with a bioactive composition incorporated therein or thereon and a mammalian cell (e.g., fibroblast) bound to the device. A mammalian tissue is contacted with the device. The scaffold composition temporally controls egress of the cell and the bioactive composition spatially or directionally regulates egress of the cell. In another example, the device that is provided contains a scaffold composition with a bioactive composition incorporated therein or thereon and a mammalian cell immobilized within the device. In the latter case, the cell remains immobilized within the device, and the scaffold composition temporally controls egress of a progeny cell of the immobilized cell and the bioactive composition spatially regulates egress of the progeny cells.

In some cases, the cells (e.g., fibroblasts) remain resident in the device for a period of time, e.g., minutes; 0.2. 0.5, 1, 2, 4, 6, 12, 24 hours; 2, 4, 6, days; weeks (1-4), months (2, 4, 6, 8, 10, 12) or years, during which the cells are exposed to structural elements and bioactive compositions that lead to proliferation of the cells, and/or a change in the activity or level of activity of the cells. The cells are contacted with or exposed to a deployment signal that induces egress of the optionally altered (re-educated or reprogrammed) cells and the cells migrate out of the device and into surrounding tissues or remote target locations.

The deployment signal is a composition such as protein, peptide, or nucleic acid. In some cases, the deployment signal is a nucleic acid molecule, e.g., a plasmid containing sequence encoding a protein that induces migration of the cell out of the device and into surrounding tissues. The deployment signal occurs when the cell encounters the plasmid in the device, the DNA becomes internalized in the cell (i.e., the cell is transfected), and the cell manufactures the gene product encoded by the DNA. In some cases, the molecule that signals deployment is an element of the device and is released from the device in delayed manner (e.g., temporally or spatially) relative to exposure of the cell to the recruitment composition.

Cells (e.g., fibroblasts) contained in the devices described herein promote regeneration of a tissue or organ (e.g., a wound) immediately adjacent to the material, or at some distant site.

In some cases, the invention described herein provides an inverse opal hydrogel scaffold device comprising a polymer matrix and a sacrificial porogen in which the porogen comprises an ionically-crosslinked polymer, a thermosensitive polymer, a thermoresponsive polymer, a pH-sensitive polymer, or a photocleavable polymer (US 2014-0178964, incorporated herein by reference). The polymer matrix is made of a durable polymer relative to the sacrificial porogen such that the polymer matrix withstands physical or chemical changes that cause porogen sacrifice. For example, polymer matrix is covalently crosslinked, withstands a change (e.g., increase) in temperature, withstands a pH change (e.g., decrease) or change in ionic strength or composition (e.g., contact with a divalent cation chelator), or withstands exposure to light (e.g., UV light).

Polynucleotides, polypeptides, or other agents are purified and/or isolated. Specifically, as used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Similarly, cell populations are substantially free of other cellular material, or culture medium. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. A purified or isolated polypeptide is free of the amino acids or sequences that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.

Similarly, by “substantially pure” is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph depicting the effect of hydrogels containing fibroblasts (originating from the skin area adjacent to an existing diabetic foot ulcer, originating from the skin of a foot of a diabetic subject without a foot ulcer, or originating from the skin of the foot of a non-diabetic subject) on wound size and time for wound closure in non-diabetic mice.

FIG. 2 is a line graph depicting the effect of hydrogels containing fibroblasts (originating from the skin area adjacent to an existing diabetic foot ulcer, originating from the skin of a foot of a diabetic subject without a foot ulcer, or originating from the skin of the foot of a non-diabetic subject) on wound size and time for wound closure in diabetic mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features compositions comprising hydrogels and fibroblasts and their use in wound healing, e.g., in diabetic subjects.

Diabetes is classified into four clinical classes: Type 1, Type 2, gestational, and diabetes due to other causes. Type 1 diabetes is caused by destruction of beta cells in the pancreas, typically leading to insulin deficiency. Type 2 diabetes is characterized by insulin resistance or hyperinsulinemia and patients often develop a progressive defect in insulin secretion. Gestational diabetes is characterized by glucose intolerance during pregnancy. Other types diabetes are due to or associated with other causes, e.g., genetic defects in insulin activity (e.g., genetic defects in the insulin receptor), pancreatic disease, hormonal diseases, genetic defects of beta cell function, or drug/chemical exposure. See, e.g., “Standards of Medical Care in Diabetes—2013.” Diabetes Care. 36.S1(2013):S11-S66; and Harris. “Classification, Diagnostic Criteria, and Screening for Diabetes.” Diabetes in America. National Institutes of Health, NIH Publication No. 95-1468. Chapter 2 (1995):15-36, incorporated herein by reference.

A subject is diagnosed as having diabetes if he or she meets one or more of following criteria: a hemoglobin A1C (A1C) level of 6.5% or higher, a fasting plasma glucose (FPG) concentration of 126 mg/dL or greater, a 2-h plasma glucose concentration of 200 mg/dL or greater during an oral glucose tolerance test (OGTT), or for subjects having symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose concentration of 200 mg/dL or greater. Thus, by “high glucose” tissue is meant tissue from a subject diagnosed with diabetes. Fasting is typically defined as no caloric intake for at least 8 hours prior to testing. The tests described herein are performed under conditions and standards generally known in the art, e.g., recommended by the World Health Organization and/or American Diabetes Association. See, e.g., “Standards of Medical Care in Diabetes—2013.” Diabetes Care. 36.S1(2013):S11-S66, incorporated herein by reference.

Because of the complications associated with diabetes (e.g., neuropathy, a weakened immune system, and narrowed arteries), even minor wounds in diabetic subjects are challenging to heal, and they either do not heal or heal slowly. Non-healing wounds progress to infection, necrosis, tissue loss, and gangrene, and eventually, amputation is necessary. As a result, more than 60% of amputations in the U.S. occurred in subjects with diabetes.

Diabetic foot ulcers are chronic, non-healing wounds on the feet of diabetic patients. These foot ulcers affect between 15-25% of Americans who have diabetes. Diabetic foot ulcers are associated with significant morbidities, a decrease in the quality of life, and often, amputation of a lower extremity.

Before the invention, previous studies showed that fibroblasts from diabetic wounds (e.g., taken from an area adjacent to a diabetic foot ulcer) were senescent and failed to grow in cultures. These characteristics were thought to be major factors in the impairment of wound healing. Surprisingly and unexpectedly, the results herein show that fibroblasts from diabetic skin near ulcers are superior in their ability to mediate wound healing, e.g., in diabetic patients. These results indicate that fibroblasts from diabetic foot ulcers retain a metabolic memory that allows them to perform better in diabetic wounds (such as those characterized by excessive inflammation) compared to fibroblasts from non-diabetic subjects. Thus, contrary to previous findings, these results show that fibroblasts taken from the area that is adjacent to diabetic foot ulcers are not senescent and can be isolated and grown in cultures.

In some examples, metabolic memory refers to the persistence of a diabetic phenotype when cells are removed from a diabetic patient (e.g., the site of a diabetic foot ulcer) and grown extensively (e.g., 4-10 passages) in a normal glucose environment (e.g., normal culture media as distinguished from high glucose culture media). See, e.g., Ceriello. Vascular Pharmacol. 57(2012):133-138; Aschner et al. Diabetes Technol. Ther. 14.1(2012):568-574 (e.g., at page S-72); Ceriello et al. J. Clin. Endocrinol. Metab. 94.2(2009):410-415; Cooper. Pediatric Diabetes. 10(2009):343-346; and Ihnat et al. Diabet. Med. 24(2007):582-586, each of which is incorporated herein by reference in its entirety.

For example, a fibroblast from a diabetic wound has metabolic memory, e.g., with an epigenetic basis or associated with an epigenetic change compared to a non-diabetic fibroblast or a fibroblast in a diabetic patient that is derived from a site located away from a diabetic wound.

A “diabetic phenotype” in this context, is defined as the expression profile of one or more protein/gene markers and/or the epigenetic alterations in cells derived from a diabetic patient (e.g., the site of a diabetic foot ulcer).

In some embodiments, epigenetic alterations include methylation of a gene.

Protein/gene markers and/or epigenetic alterations are determined by standard methods in the art, e.g., real-time polymerase chain reaction (RT-PCR) or gene expression arrays, such as microarrays.

Specifically, hydrogels developed by Mooney et al. (see, e.g., U.S. Pat. No. 8,067,237, US 2012-0100182, US 2013-0177536, US 2012-0121539, US 2013-0302396, US 2013-0331343, US 2014-0178964, US 2015-0072009, WO 12/048165, WO 12/149358, WO 12/148684, and WO 12/167230, incorporated herein by reference) were used to deliver the fibroblasts of the invention into the wounds of diabetic mice. Fibroblasts from the skin of non-diabetic subjects or diabetic subjects without foot ulceration were injected in the wounds of diabetic mice. Fibroblasts taken from the area adjacent to foot ulcers of diabetic patients performed better at wound healing than fibroblasts from the skin of non-diabetic subjects or diabetic patients without foot ulceration when injected in the wounds of diabetic mice. In contrast, fibroblasts from non-diabetic subjects performed better than fibroblasts from diabetic subjects, with or without ulceration, in non-diabetic mice.

Fibroblasts from the diabetic skin adjacent to the foot ulcer area likely retain their epigenetic changes when isolated and grown in culture. The metabolic memory that these cells retain from their original diabetic wound environment and likely underlying epigenetic changes enable them to perform better (e.g., survive longer, proliferate faster, and mediate healing) in diabetic wounds than fibroblasts from non-diabetic subjects or diabetic patients without foot ulceration. As chronic diabetic wounds are characterized by chronic inflammation, the metabolic memory (e.g., associated with epigenetic changes) in fibroblasts from diabetic foot ulcers likely facilitate improved adjustment and survival of these cells in the diabetic wound environment. Thus, the invention harnesses this metabolic memory of the fibroblasts to develop more efficient therapeutic approaches for the management of diabetic wounds (e.g., foot ulcers).

The devices of the invention provide scaffold compositions containing hydrogels that deliver these fibroblasts having superior wound-healing capabilities to a subject in need thereof. The methods and data presented herein demonstrate that these compositions are useful for the treatment of wounds, e.g., in diabetic subjects.

The invention also provides a wound product that is used for the management of diabetic foot ulceration and other chronic wounds.

In some embodiments, the invention provides a device containing a hydrogel, a fibroblast, and a stem cell for use in wound healing therapy. For example, the composition is used with induced pluripotent stem cell (iPSC) technologies to generate new stem cells for wound therapy. A stem cell is an undifferentiated cell that differentiates into a mature functional tissue specific cell upon contact with appropriate microenvironment, e.g., growth factors and other differentiating agents. In some cases, the devices described herein represent such a microenvironment. Each device constitutes a factory that attracts/accepts, reproduces, sustains, educates, and sends forth to surrounding bodily tissues tissue-specific cells that are capable of colonizing and regenerating damaged tissue. In some examples, the wound repair potency of foot ulcer fibroblasts is enhanced following their reprogramming to induced pluripotent stem cells (iPSC), e.g., in ways that enhance the cells' repair-promoting functions, e.g., functions that are mediated by epigenetic control.

Hydrogel structures are seeded with one or more populations of purified or isolated cells (e.g., isolated fibroblasts). The term “isolated” used in reference to a cell type, e.g., a fibroblast, means that the cell is substantially free of other cell types or cellular material with which it naturally occurs. For example, a sample of cells of a particular tissue type or phenotype is “substantially pure” when it is at least 60% of the cell population. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% or 100%, of the cell population. Purity is measured by any appropriate standard method, for example, by fluorescence-activated cell sorting (FACS). Optionally, the device is seeded with two or more substantially pure populations of cells. The populations are spatially or physically separated, e.g., one population is encapsulated, or the cells are allowed to come into with one another. The scaffold composition or structural support not only provides a surface upon which cells are seeded/attached but indirectly affects production/education of cell populations by housing a second (third, or several) cell population(s) with which a first population of cells associates (cell-cell adhesion).

The scaffolds compositions are seeded in vitro or in vivo. For example, scaffolds compositions are seeded by incubating the structure in a solution containing the cells. Alternatively, cells are injected/titrated into the scaffold composition or recruited to migrate into the device. In yet another example, the scaffold composition is built in stages with each layer of the multicomponent scaffold being seeded prior to laying down of another layer or before adherences of another pre-formed component. Different cell types, e.g., stem vs. differentiated, support vs. therapeutic, are optionally co-resident in the scaffold housing. Cells optionally vary in phenotype, e.g., differentiation state, activation state, metabolic state, or functional state. In general scaffolds of the invention may comprise any cell population competent to participate in regeneration, replacement or repair of a target tissue or organ. For example, the cells are fibroblasts for use in wound healing.

Cells are optionally genetically manipulated by the introduction of exogenous genetic sequences or the inactivation or modification of endogenous sequences. For example, recombinant genes are introduced to cause the cells to make proteins that are otherwise lacking in the host or target tissue. Production of scarce but desirable proteins (in the context of certain tissues) is augmented by transplanting genetically engineered cells. Cells used to seed the scaffold are capable of degrading the scaffold matrix over a desired period time in order to migrate through and out of the scaffold matrix. Scaffold matrices are selected such that they are susceptible to degradation by certain cell types seeded within the matrix. For example, scaffold materials and cells are selected and designed such that all or some of the cells seeded within the scaffold compositions require a certain desired period of time degrade the scaffold matrix sufficiently to migrate through it and reach the surrounding tissue. The delay in the release of the cells to the surrounding tissue is controlled by varying the composition of the scaffold, to allow optimal time to signal the cells to multiply, differentiate, or achieve various phenotypes. General mammalian cell culture techniques, cell lines, and cell culture systems are described in Doyle, A., Griffiths, J. B., Newell, D. G., (eds.) Cell and Tissue Culture: Laboratory Procedures, Wiley, 1998, the contents of which are incorporated herein by reference.

Cells secrete enzymes that degrade the material of the scaffold composition, thereby controlling the rate at which cells exit the scaffold. For example, migrating cells typically secrete collagenases and plasmin to degrade their matrix and allow cell movement. The rate of cells exiting may thus be regulated by controlling the density and susceptibility to these enzymes of oligopeptides used as either cross-links in the material or as components of the main chains. Certain materials are degraded in a preprogrammed manner independent of cell action (e.g. hydrolytic degradation of poly(lactide-co glyolide) as a degradable scaffold matrix. The scaffold compositions may be prepared such that the degradation time may be controlled by using a mixture of degradable components in proportions to achieve a desired degradation rate. Alternatively, the cells themselves aid in the degradation. For example, scaffold compositions are sensitive to degradation by materials secreted by the cells themselves that are seeded within the scaffold matrix. One example of this is the use of metalloproteinase (MMP)-sensitive substrate in the scaffold matrix; cells exit when the seeded cells have secreted sufficient MMP to begin degradation of the matrix.

Cells incubated in the scaffold composition are educated and induced to migrate out of the scaffold to directly affect a target tissue, e.g., and injured tissue site. For example, stromal vascular cells and smooth muscle cells are useful in sheetlike structures are used for repair of vessel-like structures such as blood vessels or layers of the body cavity. Such structures are used to repair abdominal wall injuries or defects such as gastroschisis. Similarly, sheetlike scaffold compositions seeded with dermal stem cells and/or keratinocytes are used in bandages or wound dressings for regeneration of dermal tissue.

Scaffold Compositions and Architecture

Components of the scaffold compositions are organized in a variety of geometric shapes (e.g., beads, pellets), niches, planar layers (e.g., thin sheets). For example, multicomponent scaffold compositions are constructed in concentric layers each of which is characterized by different physical qualities (% polymer, % crosslinking of polymer, chemical composition of scaffold, pore size, porosity, and pore architecture, stiffness, toughness, ductility, viscoelasticity, and or composition of bioactive substances such as growth factors, homing/migration factors, differentiation factors. Each niche has a specific effect on a cell population, e.g., promoting or inhibiting a specific cellular function, proliferation, differentiation, elaboration of secreted factors or enzymes, or migration. Cells incubated in the scaffold composition are educated and induced to migrate out of the scaffold to directly affect a target tissue, e.g., and injured tissue site. For example, stromal vascular cells and smooth muscle cells are useful in sheetlike structures are used for repair of vessel-like structures such as blood vessels or layers of the body cavity. For example, such structures are used to repair abdominal wall injuries or defects such as gastroschisis. Similarly, sheetlike scaffold compostions seeded with dermal stem cells and/or keratinocytes are used in bandages or wound dressings for regeneration of dermal tissue. The device is placed or transplanted on or next to a target tissue, in a protected location in the body, next to blood vessels, or outside the body as in the case of an external wound dressing. Devices are introduced into or onto a bodily tissue using a variety of known methods and tools, e.g., spoon, tweezers or graspers, hypodermic needle, endoscopic manipulator, endo- or trans-vascular-catheter, stereotaxic needle, snake device, organ-surface-crawling robot (United States Patent Application 20050154376; Ota et al., 2006, Innovations 1:227-231), minimally invasive surgical devices, surgical implantation tools, and transdermal patches. Devices can also be assembled in place, for example by senquentially injecting or inserting matrix materials. Scaffold devices are optionally recharged with cells or with bioactive compounds, e.g., by sequential injection or spraying of substances such as growth factors or differentiation factors.

A scaffold or scaffold device is the physical structure upon which or into which cells associate or attach, and a scaffold composition is the material from which the structure is made. For example, scaffold compositions include biodegradable or permanent materials such as those listed below. The mechanical characteristics of the scaffold composition vary according to the application or tissue type for which regeneration is sought. It is biodegradable (e.g., collagen, alginates, polysaccharides, polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide) (PLA), or poly(lactide-co-glycolide) (PLGA) or permanent (e.g., silk). In the case of biodegradable structures, the composition is degraded by physical or chemical action, e.g., level of hydration, heat or ion exchange or by cellular action, e.g., elaboration of enzyme, peptides, or other compounds by nearby or resident cells. The consistency varies from a soft/pliable (e.g., a gel) to glassy, rubbery, brittle, tough, elastic, stiff. The structures contain pores, which are nanoporous, microporous, or macroporous, and the pattern of the pores is optionally homogeneous, heterogenous, aligned, repeating, or random.

Alginates are versatile polysaccharide based polymers that may be formulated for specific applications by controlling the molecular weight, rate of degradation and method of scaffold formation. Coupling reactions can be used to covalently attach bioactive epitopes, such as the cell adhesion sequence RGD to the polymer backbone. Alginate polymers are formed into a variety of scaffold types. Injectable hydrogels can be formed from low MW alginate solutions upon addition of a cross-linking agents, such as calcium ions, while macroporous scaffolds are formed by lyophilization of high MW alginate discs. Differences in scaffold formulation control the kinetics of scaffold degradation. Release rates of morphogens or other bioactive substances from alginate scaffolds is controlled by scaffold formulation to present morphogens in a spatially and temporally controlled manner. This controlled release not only eliminates systemic side effects and the need for multiple injections, but can be used to create a microenvironment that activates host cells at the implant site and transplanted cells seeded onto a scaffold composition.

The scaffold composition comprises a biocompatible polymer matrix that is optionally biodegradable in whole or in part. A hydrogel is one example of a suitable polymer matrix material. Examples of materials which can form hydrogels include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids such as polypeptides particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon.-caprolactone, polyanhydrides; polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers of the above, including graft copolymers.

One preferred scaffold composition includes an RGD-modified alginate. Another preferred scaffold composition a macroporous poly-lactide-co-glycolide (PLG).

In other embodiments, scaffold compositions comprise a non-biodegradable material. Exemplary non-biodegradable materials include, but are not limited to, metal, plastic polymer, or silk polymer. Moreover, scaffold compositions are composed of a biocompatible material. This biocompatible material is non-toxic or non-immunogenic.

The scaffold compostions are fabricated from a variety of synthetic polymers and naturally-occurring polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels. One preferred material for the hydrogel is alginate or modified alginate material. Alginate molecules are comprised of (1-4)-linked β-D-mannuronic acid (M units) and α L-guluronic acid (G units) monomers, which can vary in proportion and sequential distribution along the polymer chain. Alginate polysaccharides are polyelectrolyte systems which have a strong affinity for divalent cations (e.g. Ca⁺², Mg⁺², Ba⁺²) and form stable hydrogels when exposed to these molecules. See Martinsen A., et al., Biotech. & Bioeng., 33 (1989) 79-89.) For example, calcium cross-linked alginate hydrogels are useful for dental applications, wound dressings chondrocyte transplantation and as a matrix for other cell types.

An exemplary device utilizes an alginate or other polysaccharide of a relatively low molecular weight, preferably of size which, after dissolution, is at the renal threshold for clearance by humans, e.g., the alginate or polysaccharide is reduced to a molecular weight of 1000 to 80,000 daltons. Prefereably, the molecular mass is 1000 to 60,000 daltons, particularly preferably 1000 to 50,000 daltons. It is also useful to use an alginate material of high guluronate content since the guluronate units, as opposed to the mannuronate units, provide sites for ionic crosslinking through divalent cations to gel the polymer. U.S. Pat. No. 6,642,363, incorporated herein by reference discloses methods for making and using polymers containing polysachharides such as alginates or modified alginates that are particularly useful for cell transplantation and tissue engineering applications.

Useful polysaccharides other than alginates include agarose and microbial polysaccharides such as those listed in the table below.

Polysaccharide Scaffold Compositions

Polymers^(a) Structure Fungal Pullulan (N) 1,4-; 1,6-α-D-Glucan Scleroglucan (N) 1,3; 1,6-α-D-Glucan Chitin (N) 1,4-β-D-Acetyl Glucosamine Chitosan (C) 1,4-β.-D-N-Glucosamine Elsinan (N) 1,4-; 1,3-α-D-Glucan Bacterial Xanthan gum (A) 1,4-β.-D-Glucan with D-mannose; D-glucuronic Acid as side groups Curdlan (N) 1,3-β.-D-Glucan (with branching) Dextran (N) 1,6-α-D-Glucan with some 1,2; 1,3-; 1,4-α-linkages Gellan (A) 1,4-β.-D-Glucan with rhamose, D-glucuronic acid Levan (N) 2,6-β-D-Fructan with some β-2,1-branching Emulsan (A) Lipoheteropolysaccharide Cellulose (N) 1,4-β-D-Glucan ^(a)N—neutral, A = anionic and C = cationic.

The scaffold compositions of the invention are porous or non-porous. For example, the scaffold compositions are nanoporous having a diameter of less than about 10 nm; microporous wherein the diameter of the pores are preferably in the range of about 100 nm-20 μm; or macroporous wherein the diameter of the pores are greater than about 20 μm, more preferably greater than about 100 μm and even more preferably greater than about 400 μm. In one example, the scaffold composition is macroporous with aligned pores of about 400-500 μm in diameter. The preparation of polymer matrices having the desired pore sizes and pore alignments are described in the Examples. Other methods of preparing porous hydrogel products are known in the art. (U.S. Pat. No. 6,511,650 incorporated herein by reference).

Scaffold compositions of the present invention contain an external surface. Scaffold compositions of the present invention alternatively, or in addition, contain an internal surface. External or internal surfaces of the scaffold compositions are solid or porous. Pore size is less than about 10 nm, in the range of about 100 nm-20 μm in diameter, or greater than about 20 μm.

Scaffold compositions of the present invention comprise one or more compartments.

The scaffold composition regulates migration of fibroblasts through the physical or chemical characteristics of the scaffold itself. For example, the scaffold composition is differentially permeable, allowing cell migration only in certain physical areas of the scaffold. The permeability of the scaffold composition is regulated, for example, by selecting or engineering a material for greater or smaller pore size, density, polymer cross-linking, stiffness, toughness, ductility, or viscoelascticity. The scaffold composition contains physical channels or paths through which cells can move more easily towards a targeted area of egress of the device or of a compartment within the device. The scaffold composition is optionally organized into compartments or layers, each with a different permeability, so that the time required for a cell to move through the device is precisely and predictably controlled. Migration is also regulated by the degradation, de- or re-hydration, oxygenation, chemical or pH alteration, or ongoing self-assembly of the scaffold composition.

Bioactive Compositions

The device includes one or more bioactive compositions. Bioactive compositions are purified naturally-occurring, synthetically produced, or recombinant compounds, e.g., polypeptides, nucleic acids, small molecules, or other agents. The compositions described herein are purified. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

Optionally, bioactive compositions are covalently or non-covalently linked to the scaffold composition. Bioactive compositions comprise an element, either covalently or non-covalently bonded to the surface of the scaffold composition, that aids in the wound healing mediated by the fibroblasts. For example, the bioactive composition promote the survival and/or proliferation of fibroblasts. In other cases, the bioactive composition reduces inflammation, e.g., at the site of the wound. In some cases, the second bioactive composition is covalently linked to the scaffold composition, keeping the composition relatively immobilized in or on the scaffold composition. In other cases, the second bioactive composition is noncovalently associated with the scaffold. Noncovalent bonds are generally one to three orders of magnitude weaker than covalent bonds permitting diffusion of the factor out of the scaffold and into surrounding tissues. Noncovalent bonds include electrostatic, hydrogen, van der Waals, π aromatic, and hydrophobic.

The bioactive composition alters a cell function or behavior, e.g., level of differentiation, state of activation, motility, or gene expression, of a cell. For example, at least one cell adhesion molecule is incorporated into or onto the polymer matrix. Such molecules are incorporated into the polymer matrix prior to polymerization of the matrix or after polymerization of the matrix. Examples of cell adhesion molecules include but are not limited to peptides, proteins and polysaccharides. More specifically, cell adhesion molecules include fibronectin, laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix protein, fibronogen, fibrin, fibulin, mucins, entactin, osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin, versican, von Willebrand Factor, polysaccharide heparin sulfate, connexins, collagen, RGD (Arg-Gly-Asp) and YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides and cyclic peptides, glycosaminoglycans (GAGs), hyaluronic acid (HA), condroitin-6-sulfate, integrin ligands, selectins, cadherins and members of the immunoglobulin superfamily. Other examples include neural cell adhesion molecules (NCAMs), intercellular adhesion molecules (ICAMs), vascular cell adhesion molecule (VCAM-1), platelet-endothelial cell adhesion molecule (PECAM-1), L1, and CHL1.

Examples of some of these molecules and their function are shown in the following table.

ECM Proteins and Peptides and Role in Cell Function

Seq. Protein Sequence ID No: Role Fibronectin RGDS 7 Adhesion LDV Adhesion REDV 8 Adhesion Vitronectin RGDV 9 Adhesion Laminin A LRGDN 10 Adhesion IKVAV 11 Neurite extension Laminin B1 YIGSR 12 Adhesion of many cells, via 67 kD laminin receptor PDSGR 13 Adhesion Laminin B2 RNIAEIIKDA 14 Neurite extension Collagen 1 RGDT 15 Adhesion of most cells DGEA 16 Adhesion of platelets, other cells Thrombospondin RGD Adhesion of most cells VTXG 17 Adhesion of platelets Hubbell, J A (1995): Biomaterials in tissue engineering. Bio/Technology 13:565-576. One-letter abbreviations of amino acids are used, X stands for any amino acid. Additional examples of suitable cell adhesion molecules are shown below. Amino Acid Sequences Specific for Proteoglycan Binding from Extracellular Matrix Proteins

SEQUENCE SEQ.ID.NO. PROTEIN XBBXBX* 18 Consensus sequence PRRARV 19 Fibronectin YEKPGSPPREVVPRPRPGV 20 Fibronectin RPSLAKKQRFRHRNRKGYRSQ 21 Vitronectin RGHSRGR rIQNLLKITNLRIKFVK 22 Laminin

Particularly preferred cell adhesion molecules are peptides or cyclic peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD) which is known as a cell attachment ligand and found in various natural extracellular matrix molecules. A polymer matrix with such a modification provides cell adhesion properties to the scaffold composition, and sustains long-term survival of mammalian cell systems, as well as supporting cell growth and differentiation.

Coupling of the cell adhesion molecules to the polymer matrix is accomplished using synthetic methods which are in general known to one of ordinary skill in the art and are described in the examples. Approaches to coupling of peptides to polymers are discussed in Hirano and Mooney, Advanced Materials, p.17-25 (2004). Other useful bonding chemistries include those discussed in Hermanson, Bioconjugate Techniques, p. 152-185 (1996), particularly by use of carbodiimide couplers, DCC and DIC (Woodward's Reagent K). Since many of the cell adhesion molecules are peptides, they contain a terminal amine group for such bonding. The amide bond formation is preferably catalyzed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which is a water soluble enzyme commonly used in peptide synthesis. The density of cell adhesion ligands, a critical regulator of cellular phenotype following adhesion to a biomaterial. (Massia and Hubbell, J. Cell Biol. 114:1089-1100, 1991; Mooney et al., J. Cell Phys. 151:497-505, 1992; and Hansen et al., Mol. Biol. Cell 5:967-975, 1994) can be readily varied over a 5-order of magnitude density range.

Device Construction

The scaffold structure is constructed out of a number of different rigid, semi-rigid, flexible, gel, self-assembling, liquid crystalline, or fluid compositions such as peptide polymers, polysaccharides, synthetic polymers, hydrogel materials, ceramics (e.g., calcium phosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans, metals and metal alloys. The compositions are assembled into cell scaffold structures using methods known in the art, e.g., injection molding, lyophillization of preformed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, gas foaming, fiber forming/processing, particulate leaching or a combination thereof. The assembled devices are then implanted or administered to the body of an individual to be treated.

The device is assembled in vivo in several ways. The scaffold composition is made from a gelling material, which is introduced into the body in its ungelled form where it gells in situ. Exemplary methods of delivering device components to a site at which assembly occurs include injection through a needle or other extrusion tool, spraying, painting, or methods of deposit at a tissue site, e.g., delivery using an application device inserted through a cannula. In one example, the ungelled or unformed scaffold material is mixed with bioactive substances and cells prior to introduction into the body or while it is introduced. The resultant in vivo/in situ assembled scaffold contains a mixture of these substances and cells.

In situ assembly of the scaffold composition occurs as a result of spontaneous association of polymers or from synergistically or chemically catalyzed polymerization. Synergistic or chemical catalysis is initiated by a number of endogenous factors or conditions at or near the assembly site, e.g., body temperature, ions or pH in the body, or by exogenous factors or conditions supplied by the operator to the assembly site, e.g., photons, heat, electrical, sound, or other radiation directed at the ungelled material after it has been introduced. The energy is directed at the scaffold material by a radiation beam or through a heat or light conductor, such as a wire or fiber optic cable or an ultrasonic transducer. Alternatively, a shear-thinning material, such as an ampliphile, is used which re-cross links after the shear force exerted upon it, for example by its passage through a needle, has been relieved.

Suitable hydrogels for both in vivo and ex vivo assembly of scaffold devices are well known in the art and described, e.g., in Lee et al., 2001, Chem. Rev. 7:1869-1879. The peptide amphiphile approach to self-assembly assembly is described, e.g., in Hartgerink et al., 2002, Proc. Natl. Acad. Sci. U. S. A. 99:5133-5138. A method for reversible gellation following shear thinning is exemplied in Lee et al., 2003, Adv. Mat. 15:1828-1832

A multiple compartment device is assembled in vivo by applying sequential layers of similarly or differentially doped gel or other scaffold material to the target site. For example, the device is formed by sequentially injecting the next, inner layer into the center of the previously injected material using a needle, forming concentric spheroids. Non-concentric compartments are formed by injecting material into different locations in a previously injected layer. A multi-headed injection device extrudes compartments in parallel and simultaneously. The layers are made of similar or different scaffolding compositions differentially doped with bioactive substances and different cell types. Alternatively, compartments self-organize based on their hydro-philic/phobic characteristics or on secondary interactions within each compartment.

Growth Factors and Incorporation of Compositions Into/Onto a Scaffold Device

Bioactive substances that influence growth, development, movement, and other cellular functions are introduced into or onto the scaffold structures. Such substances include BMP, bone morphogenetic protein; ECM, extracellular matrix proteins or fragments thereof; EGF, epidermal growth factor; FGF-2, fibroblast growth factor 2; NGF, nerve growth factor; PDGF, platelet-derived growth factor; PIGF, placental growth factor; TGF, transforming growth factor, and VEGF, vascular endothelial growth factor, phosphatase inhibitors. Cell-cell adhesion molecules (cadherins, integrins, ALCAM, NCAM, proteases) are optionally added to the scaffold composition. Exemplary growth factors and ligands are provided in the tables below. Preferably, the growth factor/ligand comprises VEGF, PDGF, HGF, and/or RGD.

Growth Factors Used for Angiogenesis

Growth factor Abbreviation Relevant activities Vascular endothelial VEGF Migration, proliferation and growth factor survival of ECs Basic fibroblast bFGF-2 Migration, proliferation and growth factor survival of ECs and many other cell types Platelet-derived PDGF Promotes the maturation of growth factor blood vessels by the recruitment of smooth muscle cells Angiopoietin-1 Ang-1 Strengthens EC-smooth muscle cell interaction Angiopoietin-2 Ang-2 Weakens EC-smooth muscle cell interaction Placental growth PIGF Stimulates angiogenesis factor Transforming growth TGF Stabilizes new blood vessels factor by promoting matrix deposition

Growth Factors Used for Bone Regeneration

Growth factor Abbreviation Relevant activities Transforming growth TGF-β Proliferation and factor-β differentiation of bone-forming cells Bone morphogenetic BMP Differentiation of protein bone-forming cells Insulin-like growth IGF-1 Stimulates proliferation factor of osteoblasts and the synthesis of bone matrix Fibroblast growth FGF-2 Proliferation of osteoblasts factor-2 Platelet-derived PDGF Proliferation of osteoblasts growth factor

Growth Factors Used for Wound Healing

Growth Factor Abbreviation Relevant activities Platelet-derived PDGF Active in all stages of growth factor healing process Epidermal growth EGF Mitogenic for keratinocytes factor Transforming growth TGF-β Promotes keratinocyte factor-β migration, ECM synthesis and remodeling, and differentiation of epithelial cells Fibroblast growth FGF General stimulant for wound factor healing

Growth Factors Used for Tissue-Engineering

Moleular Representative supplier Growth factor Abbreviation weight (kDa) Relevant activities of rH growth factor Epidermal growth EGF 6.2 Proliferation of epithelial, mesenchymal, and PeproTech Inc. (Rocky factor fibroblast cells Hill, NJ, USA) Platelet-derived PDGF-AA 28.5 Proliferation and chemoattractant agent for PeproTech Inc. growth factor PDGF-AB 25.5 smooth muscle cells; extracellular matrix PDGF-BB 24.3 synthesis and deposition Transforming TFG-α 5.5 Migration and proliferation of keratinocytes; PeproTech Inc. growth factor-α extracellular matrix synthesis and deposition Transforming TGF-β 25.0 Proliferation and differentiation of bone PeproTech Inc. growth factor-β forming cells; chemoattractant for fibroblasts Bone morphogenetic BMP-2 26.0 Differentiation and migration of bone Cell Sciences Inc. protein BMP-7 31.5 forming cells (Norwood, MA, USA) Basic fibroblast bFGF/FGF-2 17.2 Proliferation of fibroblasts and initiation of PeproTech Inc. growth factor angiogenesis Vascular endothelial VEGF₁₆₅ 38.2 Migration, proliferation, and survival of PeproTech Inc. growth factor endothelial cells rH, recombinant human

Immobilized Ligands Used in Tissue Engineering

Immobilized ECM molecule ligand* source Application RGD Multiple ECM Enhance bone and cartilage tissue molecules, formation in vitro and in vivo including Regulate neurite outgrowth in fibronectin, vitro and in vivo vitronectin, Promote myoblast adhesion, laminin, proliferation and differentiation collagen and Enhance endothelial cell thrombospondin adhesion and proliferation IKVAV YIGSR Laminin Regulate neurite outgrowth in RNIAEIIKDI vitro and in vivo Recombinant Fibronectin Promote formulation of focal fibronectin contacts in pre-osteoblasts fragment (FNIII₇₋₁₀) Ac-GCRDGPQ- Common MMP Encourage cell-mediated GIWGQDRCG substrates, proteolytic degradation, (e.g. collagen, remodeling and bone fibronectin, regeneration (with RGD and laminin) BMP-2 presentation) in vivo *Sequences are given in single-letter amino acid code. MMP, matrix metalloproteinase.

The release profiles of bioactive substances from scaffold devices is controlled by both factor diffusion and polymer degradation, the dose of the factor loaded in the system, and the

composition of the polymer. Similarly, the range of action (tissue distribution) and duration of action, or spatiotemporal gradients of the released factors are regulated by these variables. The diffusion and degradation of the factors in the tissue of interest is optionally regulated by chemically modifying the factors (e.g., PEGylating growth factors). In both cases, the time frame of release determines the time over which effective cell delivery by the device is desired.

Carrier systems for tissue regeneration are described in the table below.

Polymeric Carriers Used to Deliver Various Growth Factors and the Type of Tissues Regenerated

Growth factor Carrier Tissue regenerated EGF Gelatin Dermis PET suture Tendon PVA sponge Dermis PDGF Chitosan-PLLA scaffold Craniofacial bone CMC gel Dermis Fibrin Ligament Porous HA Long Bone TGF-β Alginate Cartilage PLA Long Bone CaP-titanium mesh Craniofacial bone Polyoxamer; PEO gel Dermis rhBMP-2 Collagen sponge Long bone Craniofacial bone HA-TCP granules Spinal bone HA-collagen Long bone PLA-DX-PEG Ectopic and hip bone rHBMP-7 HA Spinal bone Collagen-CMC Spinal bone Porous HA Craniofacial bone bFGF Chitosan Dermis Heparin-alginate Blood vessels EVAc microspheres Blood vessels Fibrin matrices Blood vessels VEGF PLG scaffold Blood vessels PLG scaffold Blood vessels PLG microspheres Blood vessels Fibrin mesh Blood vessels Abbreviations: PET, poly (ethylene terepthalate); PVA, polyvinyl alcohol; PLLA, poly(L-lactic acid); CMC, carboxymethylcellulose; HA, hydroxyapatite; PLA, poly(D,L-lactic acid); CaP, calcium phosphate; PEO, poly (ethylene oxide); TCP, tricalcium phosphate; PEG, poly(ethylene glycol); -DX-, -p-dioxanone-; EVAc, ethylene vinyl acetate; PLG, poly(lactide-co-glycolide).

The bioactive substances are added to the scaffold compositions using known methods including surface absorption, physical immobilization, e.g., using a phase change to entrap the substance in the scaffold material. For example, a growth factor is mixed with the scaffold composition while it is in an aqueous or liquid phase, and after a change in environmental conditions (e.g., pH, temperature, ion concentration), the liquid gels or solidifies thereby entrapping the bioactive substance. Alternatively, covalent coupling, e.g., using alkylating or acylating agents, is used to provide a stable, longterm presentation of a bioactive substance on the scaffold in a defined conformation. Exemplary reagents for covalent coupling of such substances are provided in the table below.

Methods to Covalently Couple Peptides/Proteins to Polymers

Functional Group of Reacting groups on Polymer Coupling reagents and cross-linker proteins/peptides —OH Cyanogen bromide (CNBr) —NH₂ Cyanuric chloride 4-(4,6-Dimethoxy-1,3,5-triazin-2- yl)-4-methyl-morpholinium chloride (DMT-MM) —NH₂ Diisocyanate compounds —NH₂ Diisothoncyanate compounds —OH Glutaraldehyde Succinic anhydride —NH₂ Nitrous Acid —NH₂ Hydrazine + nitrous acid —SH —Ph—OH —NH₂ Carbodiimide compounds (e.g., —COOH EDC, DCC)[a] DMT-MM —COOH Thionyl chloride —NH₂ N-hydroxysuccinimide N-hydroxysulfosuccinimide + EDC —SH Disulfide compound —SH [a]EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; DCC: dicyclohexylcarbodiimide

Bioactive substances are capable of inducing migration of the transplanted cells and their progeny out of the polymer matrix. Other preferred bioactive substances are capable of maintaining cell viability, promoting cell proliferation or preventing premature terminal differentiation of the transplanted cells. Such bioactive substances are used alone or in combination to achieve the desired result.

Bioactive substances suitable for use in the present invention include, but are not limited to: growth factors, hormones, neurotransmitters, neurotransmitter or growth factor receptors, interferons, interleukins, chemokines, cytokines, colony stimulating factors, chemotactic factors, MMP-sensitive substrate, extracellular matrix components; such as growth hormone, parathyroid hormone (PTH), bone morphogenetic protein (BMP), transforming growth factor-α (TGF-α), TGF-β1, TGF-β2, fibroblast growth factor (FGF), granulocyte/macrophage colony stimulating factor (GMCSF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), insulin-like growth factor (IGF), scatter factor/hepatocyte growth factor (HGF), fibrin, collagen, fibronectin, vitronectin, hyaluronic acid, an RGD-containing peptide or polypeptide, an angiopoietin and vascular endothelial cell growth factor (VEGF). Splice variants of any of the above mentioned proteins, and small molecule agonists or antagonists thereof that may be used advantageously to alter the local balance of pro and anti-migration and differentiation signals are also contemplated herein.

Examples of cytokines as mentioned above include, but are not limited to IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interferon-γ (γ-IFN), IFN-α, tumor necrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40 ligand.

Suitable bioactive substances useful in accordance with the invention also include but are not limited to DNA molecules, RNA molecules, antisense nucleic acids, ribozymes, plasmids, expression vectors, marker proteins, transcription or elongation factors, cell cycle control proteins, kinases, phosphatases, DNA repair proteins, oncogenes, tumor suppressors, angiogenic proteins, anti-angiogenic proteins, cell surface receptors, accessory signaling molecules, transport proteins, enzymes, anti-bacterial agents, anti-viral agents, antigens, immunogens, apoptosis-inducing agents, anti-apoptosis agents, and cytotoxins.

For some applications, the scaffold compositions of the invention include at least one cell growth factor that prevents premature terminal differentiation of the transplanted cells in the polymer matrix and induces migration of the transplanted cells and their progeny out of the polymer matrix. Cell growth factors are incorporated into the polymer matrix prior to polymerization of fabrication or may be coupled to the polymer matrix after polymerization. The choice of growth factor will depend upon the type of cells and the influence of a particular growth factor on those cells such that the cells are directed to bypass their normal tendency to differentiate, and remain in a proliferative phase until a sufficient number of cells is attained to regenerate the targeted tissue and for the cells to have also migrated from the scaffold.

Scaffold compositions of the invention optionally comprise at least one non-viral gene therapy vector such that either the transplanted cells or host cells in the vicinity of the implant would take up and express gene that lead to local availability of the desired factor for a desirable time frame. Such non-viral vectors include, but are not limited to, cationic lipids, polymers, targeting proteins, and calcium phosphate.

For regeneration of muscular tissue, the cells seeded in the scaffold composition are myoblasts and the preferred combination of growth factors is HGF and FGF2. FGF2 is particularly useful in preventing the premature differentiation of the transplanted cells, while HGF induces migration of the cells from the scaffold composition. The incorporation of the two growth factors significantly increased the viability and migration of the seeded myoblasts as discussed below.

The biocompatible scaffold compositions of the invention are useful in a broad range of in vivo and in vitro regenerative medicine and tissue engineering. Devices are designed and manufactured for a wide variety of injuries, diseases, conditions and cell therapies, and delivered to the treatment location using surgical, endoscopic, endovascular, and other techniques. The devices degrade and resorb after the treatment is successfully completed or remain in place permantly or semi-permanently. Cells are seeded ex vivo into the scaffold composition with autologous or allogeneic cells. The devices are particularly useful in regenerating heart tissue (ischemia lesions and scarring), dermal tissue (scarring, ulcers, burns), CNS tissue (spinal cord injury, MS, ALS, dopamine shortage), and for skeletal-muscle system repairs (tendons, ligaments, discs, post-surgical, hernias)

The invention also provides a method for treating a patient in need of wound healing and/or tissue regeneration, replacement or repair (e.g., at or around the site of a wound, such as a diabetic wound) comprises the step implanting a scaffold composition in or near the tissue in need of regeneration, repair or replacement. This method for treating a patient in need of wound healing involves implanting in the patient a biocompatible scaffold containing a macroporous, polymer matrix having at least a population of fibroblasts capable of mediating wound healing transplanted within the polymer matrix; and optionally at least one cell growth inductive factor that prevents terminal differentiation of the transplanted cells in the polymer matrix and induces migration of the transplanted cells and their progeny out of the polymer matrix. For example, the cell growth inductive factor(s) is a combination of HGF and FGF2.

The devices are useful to treat acute and chronic tissue disease or defects (e.g., non-healing or slow-healing wounds) in humans as well as animals such as dogs, cats, horses, and other domesticated and wild animals.

The devices increase the efficacy of stem and transgenic cell therapies, and the devices are tailored to suit each clinical problem with the appropriate choice of scaffold composition, pore size, bioactive substance(s) and cell types. The device solves the major problem of efficiently integrating therapeutic cells into target tissue. Physicians place the device near the site requiring therapy or regeneration, where it delivers a flow of cells (e.g., fibroblasts) to the target site. Unlike traditional scaffold compositions, the scaffold in the device exports cells such as fibroblasts after they have incubated, replicated and matured inside the device. The device has shown 20X+improvements in viable cell delivery and tissue re-growth for damaged skeletal muscle. By matching its design to the specific cell type biochemistry, the device causes an extended stream of matured cells to migrate into the target tissue (e.g., the site of or area around a wound).

The devices offer several advantages over other scaffold systems. Maximum therapeutic efficacy is achieved, because cells are delivered in prime condition at the right time in the right quantities directly to the locus of a wound. Sustained delivery facilitates accretive integration of therapeutic cells into tissue at a desired location. The devices has been shown to be more efficient in viable cell delivery (110% for this device vs. 5% for the best alternative techniques). Thus, fewer cells are needed per treatment allowing successful therapies which might have failed at lower cell delivery rates. Lower cell numbers also permit autologous grafts, because fewer cells need to be harvested from the patient to be treated and less time is required between harvest and graft to proliferate cells in vitro. Since fewer cells are required, relative rare cells can be used. The devices also permit less expensive allogeneic grafts. Other advantages include rapid determination of the therapeutic benefit of any treatment and faster tissue growth and enhanced healing.

The invention provides a composition comprising a hydrogel and a population of fibroblasts. The hydrogel comprises pores, and the population of fibroblasts is bound to the hydrogel. For example, the population of fibroblasts is seeded into or onto the hydrogel.

In some cases, the population of fibroblasts comprises a fibroblast that is derived from a subject suffering from diabetes or a subject having a wound. For example, the wound is located in an extremity (e.g., an arm, hand, leg, or foot). In some cases, the wound is a diabetic wound. For example, the wound is an ulcer (e.g., an arm, hand, leg, or foot ulcer). Inflammation of tissues in and/or around a wound commonly occurs.

Diabetes is a chronic disease in which the body fails to properly regulate glucose metabolism. Types of diabetes include Type 1, Type 2, and gestational diabetes. Type 1 diabetes can develop at any age, but it commonly appears in children and adolescents. Type 1 diabetes is caused by a lack of sufficient insulin production to regulate glucose metabolism. Insulin is normally secreted by the beta cells of the pancreas to lower the amount of glucose in the blood. The lack of insulin production is caused by defective/damaged beta cells in the pancreas (which are cells that produce insulin). For example, damaged beta cells in Type 1 diabetics are destroyed by immune cells.

The onset of Type 2 diabetes can occur at any age. In Type 2 diabetes, the body is insulin resistant and the pancreas is unable to make enough insulin to overcome the resistance. In normal subjects, insulin leads to an uptake of glucose from the blood into cells. For example, cell types such as fat and muscle cells respond to insulin by absorbing glucose. Also, liver cells normally respond to insulin by reducing their secretion of glucose into the blood. However, when cells fail to insulin (as in Type 2 diabetes), blood glucose levels rise. Type 2 diabetes commonly occurs in overweight or obese subjects.

Symptoms and associated disorders of Type 1 and Type 2 diabetes due to the elevated blood sugar include increased thirst, frequent urination, extreme hunger, unexplained weight loss, slow-healing sores/wounds, presence of ketones in the urine, fatigue, blurred vision, blindness, high blood pressure, frequent infections, loss of kidney function, nerve damage, heart and blood vessel disease, gangrene, and ulcers (e.g., in an extremity).

Slow-healing or non-healing wounds commonly occur in diabetics. A wound includes an open wound or a closed wound. For example, an open wound occurs due to an injury to the skin, where the skin is cut, punctured, or torn. Open wounds include incisions, lacerations, abrasions, avulsions, puncture wounds, and penetration wounds. In closed wounds, the skin is not cut, punctured, or torn, but tissue under the skin is injured, e.g., from blunt force trauma. Close wounds include contusions (e.g., bruises), hematomas (e.g., caused by damage to a blood vessel that causes blood to pool under the skin), and crush injuries (e.g., caused by a large amount of force applied to a site of the body over an extended period of time).

One of the most significant complications of diabetes is chronic, non-healing wounds of an extremity, such as a foot. Due to nerve damage in the feet and legs of diabetics, small wounds/irritations often develop into chronic, non-healing wounds without the patient's awareness. Also, because of the damaged microvasculature of diabetics, such wounds take a long time to heal, if at all. A significant percentage of diabetic patients eventually develop foot ulcers, which lead to amputations if not aggressively treated. The present invention provides a method to treat diabetic wounds and/or ulcers, e.g., in an extremity, by using fibroblasts.

A fibroblast is a type of cell of connective tissue that produces collagen and the extracellular matrix (e.g., extracellular matrix proteins such as collagen, glycosaminoglycan, reticular and elastic fiber, and other glycoproteins), which serve a structural role for animal tissues. Fibroblasts are important in the process of wound healing, and tissue damage stimulates the proliferation of fibroblasts.

The process of wound healing comprises several phases: hemostasis, inflammation, proliferation, and remodeling. Upon injury (e.g., to the skin), platelets aggregate at the site of injury to from a clot in order to reduce bleeding. This process is called hemostasis. In the inflammation phase, white blood cells remove bacteria and cell debris from the wound. In the proliferation phase, angiogenesis (formation of new blood vessels by vascular endothelial cells) occurs, as does collagen deposition, tissue formation, epithelialization, and wound contraction at the site of the wound. To form tissue at the site of the wound, fibroblasts grow to form a new extracellular matrix by secreting proteins such as fibronectin and collagen. Re-epithelialization also occurs in which epithelial cells proliferate and cover the site of the wound in order to cover the newly formed tissue. In order to cause wound contraction, myofibroblasts decrease the size of the wound by contracting and bringing in the edges of the wound. In the remodeling phase, apoptosis occurs to remove unnecessary cells at the site of the wound. One or more of these phases in the process of wound healing is disrupted or delayed in non-healing/slow-healing wounds, e.g., due to diabetes, old age, or infections.

In some embodiments, the fibroblast is derived from the site of the wound or a site adjacent to the wound. For example, at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more) of the fibroblasts in the population are derived from the site of the wound or a site adjacent to the wound.

In some cases, the population of fibroblasts comprises a fibroblast that is derived from a portion of the skin of the subject. For example, at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more) of the fibroblasts in the population are derived from a portion of the skin of the subject. In some cases, the population of fibroblasts further comprises a fibroblast that is derived from healthy subject, e.g., a subject not suffering from diabetes. For example, the fibroblast is derived from the skin of the healthy subject. In addition or alternatively, the population of fibroblasts comprises a fibroblast that is derived from a subject (e.g., healthy or diseased subject) where the fibroblast is derived from a non-diseased or non-injured site on the body of the subject (e.g., from a site on the skin of the subject, where the site is not adjacent to a wound).

The subject is a mammal, e.g., a human, primate, monkey, cow, horse, pig, dog, cat, mouse, rabbit, or rat. Preferably, the subject is a human.

In some cases, the population of fibroblasts includes fibroblasts that have been cultured in vitro. For example, the population of fibroblasts includes at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more) fibroblasts that have been cultured in vitro.

For example, fibroblasts are isolated from a mammalian tissue, such as skin. Fibroblasts are separated from other cell types using conventional cell fractionation methods commonly known in the art. For example, cells are fractionated using a density gradient separation or using flow cytometry sorting. The isolated cells are at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%) fibroblasts. The isolated cells are plated in a cell culture dish or flask at a density of from 10³ to 10⁹ cells per mL of culture. The cells are then cultured in standard culture media suitable for the culture of fibroblasts, e.g., IMDM, MEM, DMEM, RPMI 1640, Alpha Medium, or McCoy's Medium. The culture media optionally contains a serum component, e.g., horse, human, fetal calf, newborn calf, or calf serum. Cells are cultured for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or more days. For example, the cells are cultured for less than 12 days. Standard culture schedules call for medium and serum to be exchanged weekly, either as a single exchange performed weekly or a one-half medium and serum exchange performed twice weekly. Preferably, the nutrient medium of the culture is replaced, preferably perfused, either continuously or periodically, at a rate of about 1 ml per ml of culture per about 24 to about 48 hour period, for cells cultured at a density of from 2×10⁶ to 1×10⁷ cells per ml. After culturing, the cells are harvested, for example using an enzyme such as trypsin, and washed to remove the growth medium. The cells are resuspended in a pharmaceutical grade electrolyte solution, for example Isolyte (B. Braun Medical Inc., Bethlehem, PA) and optionally supplemented with serum albumin.

In some embodiments, the population of fibroblasts includes a fibroblast with metabolic memory, e.g., associated with an epigenetic alteration, compared to a fibroblast derived from a subject not suffering from diabetes and/or not having a wound.

In some embodiments, a fibroblast is epigenetically altered at one or more target sites (e.g., genes) shown in Table 1 below. For example, the fibroblast (e.g., diabetic foot ulcer (DFU) fibroblast) contains an alteration (e.g., methylation) in one or more of the genes shown in Table 1 below compared to non-diabetic fibroblasts (NFF). In other examples, the fibroblast (e.g., diabetic foot ulcer (DFU) fibroblast) contains an increase or decrease (e.g., by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or greater) in methylation of one or more of the genes shown in Table 1 below compared to non-diabetic fibroblasts (NFF) and/or in methylation of one or more site within a gene shown in Table 1 compared to non-diabetic fibroblasts. For example, the fibroblast (e.g., diabetic foot ulcer (DFU) fibroblast) contains an increase or decrease by at least 2-fold in methylation of one or more of the genes shown in Table 1 below compared to non-diabetic fibroblasts (NFF) and/or in methylation of one or more site within a gene shown in Table 1 compared to non-diabetic fibroblasts. Differential methylation is determined using methods commonly available in the art, e.g., methylation assays available from IIlumina, bisulfite sequencing, and/or pyrosequencing. See, e.g., Weisenberger et al. “Comprehensive DNA Methylation Analysis on the Illumina® Infinium® Assay Platform.” Illumina, Inc. Mar. 25, 2008. Web. May 9, 2014. res.illumina.com/documents/products/appnotes/appnote_dna_methylation_analysis_infinium.pdf

To generate the information in Table 1, an Illumina-style annotation method was used. Differentially methylated probes were used to identify those sites in a gene/chromosome that contained altered methylation in DFU vs. NFF. The table below lists these differentially methylated sites and provides information regarding the functions of these sites/areas of the chromosome based on previous observations and classifications. These functions are useful for interpreting the role of (e.g., biological pathways affected by or disease states associated with) the differential methylation observed in NFF v. DFU.

The beta.median value was calculated as the median of beta across the 4 biological replicate samples in the NFF and DFU groups, respectively. Beta was calculated as beta=M/(M+U+alpha), where M is the values of the methylated probe and U is the value of the unmethylated probe and alpha is an arbitrary offset of 100. The M and U values were converted from fluorescence readings on the array.

UCSC_CpG_Islands_Name indicates the chromosomal location of a CpG island as defined by the University of California Southern California database (UCSC). If the single nucleotide interrogated by the differentially methylated probe fell within this region of the CpG island, then it was considered to be annotated to this CpG island.

The Phantom column refers to whether the differentially methylated probe fell within a promoter as defined by the FANTOM (Functional Annotation of the Mammalian genome) algorithm. See, e.g., Katayama et al. Brief Bioinform. 5.3(2004):249-258, incorporated herein by reference.

DMR refers to a differentially methylated region. The DMR column indicates whether this site has been identified to be differentially methylated in other experiments as interpreted by a consortium of epigenetics experts assembled by Illumina. This information may be from different cell types and biological contexts. The DMR information could shed light into whether this particular site is prone to methylation differences. RDMR refers to a reprogramming-specific DMR, CDMR refers to a cancer DMR, and DMR refers a differentially methylated region that is not further classified.3

The Enhancer column indicates whether this region is determined to be an enhancer of gene expression as determined by bioinformatic methods according to a consortium of epigenetics experts assembled by Illumina. TRUE means that this region has been identified as an enhancer.

The HMM_Island indicates whether the site containing the differentially methylated probe is identified as a CpG island using the hidden markov model algorithm. See, e.g., Yoon. Curr. Genomics. 10.6(2009):402-415, incorporated herein by reference. This column provides similar information as the UCSC_CpG_Island column and overall offers insight into the genomic context and potential regulatory roles of DNA methylation at this site.

The Regulatory_Feature_Name and Regulatory_Feature_Group columns provide information regarding the gene expression regulatory features of the differentially methylated site as described by the consortium of epigenetics experts assembled by 11lumina. This information is useful for interpreting the regulatory roles of the sites that are differentially methylated between DFU v. NFF.

DHS refers to DNase I hypersensitive sites, and TRUE within the DHS column means that the differentially methylated probe fell within an identified DHS site. This information is useful for elucidating the potential regulatory roles of DNA methylation at this site, as methylation within accessible DHS sites may regulate gene expression.

Throughout Table 1, NA means Not Applicable because this specific category is not representative of the context of that specific differentially methylate probe site.

TABLE 1 Differential methylation of genes in DFU versus NFF using an Illumina-style annotation method UCSC_(—) NFF. DFU. UCSC_(—) UCSC_(—) UCSC_(—) CpG_(—) Relation_(—) beta. beta. RefGene_(—) RefGene_(—) RefGene_(—) Islands_(—) to_UCSC_(—) Name chr median median Name Accession Group Name CpG_Island cg00156230 chr7:45073692 0.3 0.54 CCM2; NM_031443; Body; NA NA CCM2; NM_001029835; Body; CCM2; NM_001167935; Body; CCM2; NM_001167934; Body; CCM2 NR_030770 Body cg00264799 chr12:102848839 0.74 0.41 IGF1; NM_001111285; Body; NA NA IGF1; NM_000618; Body; IGF1; NM_001111283; Body; IGF1 NM_001111284 Body cg00290607 chr11:67383545 0.54 0.31 NA NA NA chr11:67383537- Island 67383809 cg00481216 chr2:181971175 0.44 0.66 NA NA NA NA NA cg00518941 chr2:46361964 0.53 0.74 PRKCE NM_005400 Body NA NA cg00575645 chr5:169548502 0.72 0.51 NA NA NA NA NA cg00806704 chr13:28539042 0.57 0.33 CDX2 NM_001265 Body chr13:28540356- N_Shore 28541279 cg00945409 chr10:80737665 0.81 0.45 LOC283050; NR_024431; Body; chr10:80733751- S_Shelf LOC283050; NR_024429; Body; 80734013 LOC283050 NR_015429 Body cg01230320 chr2:19549980 0.91 0.59 NA NA NA chr2:19551735- N_Shore 19551961 cg01395541 chr6:34524766 0.77 0.57 SPDEF NM_012391 TSS1500 NA NA cg01468567 chr19:49843922 0.48 0.7 TEAD2 NM_003598 3′UTR chr19:49842018- S_Shore 49842323 cg01470456 chr8:85787158 0.86 0.63 RALYL; NM_001100393; Body; NA NA RALYL; NM_001100392; Body; RALYL; NM_001100391; Body; RALYL NM_173848 Body cg01549977 chr14:65743867 0.73 0.47 NA NA NA chr14:65746329- N_Shelf 65746972 cg01632562 chr6:45629759 0.5 0.28 NA NA NA chr6:45630986- N_Shore 45631814 cg01694488 chr4:1580172 0.14 0.39 NA NA NA chr4:1580050- Island 1580455 cg01790920 chr5:3594679 0.49 0.25 IRX1 NM_024337 TSS1500 chr5:3594467- Island 3603054 cg02100397 chr19:646890 0.88 0.53 NA NA NA chr19:645890- Island 648623 cg02138953 chr10:75666279 0.64 0.35 NA NA NA NA NA cg02159489 chr17:79459563 0.09 0.5 NA NA NA chr17:79454734- S_Shelf 79455823 cg02197634 chr6:33048875 0.19 0.47 HLA- NM_002121 Body chr6:33048416- S_Shore DPB1 33048814 cg02368820 chr1:3052501 0.27 0.66 PRDM16; NM_022114; Body; NA NA PRDM16 NM_199454 Body cg02395396 chr1:52435307 0.57 0.34 RAB3B NM_002867 Body NA NA cg02527375 chr3:41724686 0.66 0.39 ULK4 NM_017886 Body NA NA cg02532518 chr16:3210066 0.56 0.34 NA NA NA chr16:3207597- S_Shore 3209413 cg02799905 chr2:206342226 0.68 0.45 PARD3B; NM_152526; Body; NA NA PARD3B; NM_057177; Body; PARD3B NM_205863 Body cg02890259 chr1:16345207 0.09 0.46 HSPB7; NM_014424; 1stExon; NA NA HSPB7 NM_014424 5′UTR cg03079497 chr17:1390554 0.14 0.35 MYO1C; NM_03375; 5′UTR; chr17:1390456- Island MYO1C NM_001080779 Body 1390786 cg03088219 chr4:176711190 0.57 0.32 GPM6A; NM_201591; Body; NA NA GPM6A; NM_005277; Body; GPM6A NM_201592 Body cg03119829 chr1:64170964 0.42 0.69 NA NA NA NA NA cg03217995 chr7:27203430 0.36 0.58 HOXA9 NM_152739 Body chr7:27203915- N_Shore 27206462 cg03301058 chr6:90007856 0.6 0.34 GABRR2 NM_002043 Body NA NA cg03487027 chr10:77159055 0.3 0.72 ZNF503 NM_032772 Body chr10:77155128- Island 77169600 cg03653601 chr16:16156039 0.36 0.65 ABCC1; NM_019862; Body; NA NA ABCC1; NM_019898; Body; ABCC1; NM_019899; Body; ABCC1; NM_004996; Body; ABCC1 NM_019900 Body cg03814093 chr4:154410006 0.45 0.65 KIAA0922; NM_015196; Body; NA NA KIAA0922 NM_001131007 Body cg03859028 chr15:99949289 0.17 0.4 NA NA NA NA NA cg04478875 chr5:142023774 0.67 0.4 FGF1; NM_000800; 5′UTR; NA NA FGF1; NM_033136; 5′UTR; FGF1; NM_001144935; 5′UTR; FGF1; NM_001144934; 5′UTR; FGF1; NR_026696; Body; FGF1 NR_026695 Body cg04500819 chr1:170376975 0.81 0.5 NA NA NA NA NA cg04506342 chr2:160463692 0.42 0.85 BAZ2B NM_013450 5′UTR NA NA cg04887066 chr12:32185731 0.57 0.31 NA NA NA NA NA cg04888234 chr1:161675579 0.85 0.48 FCRLA NM_032738 TSS1500 NA NA cg04894537 chr11:2763171 0.41 0.2 KCNQ1; NM_000218; Body; NA NA KCNQ1 NM_181798 Body cg04998634 chr19:1857004 0.13 0.39 KLF16 NM_031918 Body chr19:1856725- Island 1857443 cg05210689 chr14:100233454 0.55 0.26 NA NA NA NA NA cg05227215 chr5:139057496 0.24 0.47 CXXC5 NM_016463 5′UTR chr5:139056577- S_Shore 139056856 cg05276972 chr11:130482015 0.82 0.43 NA NA NA NA NA cg05279330 chr13:26776254 0.9 0.65 NA NA NA NA NA cg05422883 chr2:43072932 0.42 0.65 NA NA NA NA NA cg05424060 chr7:79768675 0.4 0.63 GNAI1 NM_002069 Body chr7:79763793- S_Shelf 79764889 cg05494467 chr5:140892308 0.12 0.33 PCDHGB5; NM_018925; 3′UTR; chr5:140892913- N_Shore PCDHGC3; NM_002588; 3′UTR; 140893189 PCDHGA6; NM_018919; 3′UTR; PCDHGB4; NM_003736; 3′UTR; PCDHGA8; NM_032088; 3′UTR; PCDHGA12; NM_003735; 3′UTR; PCDHGB3; NM_018924; 3′UTR; PCDHGA5; NM_018918; 3′UTR; PCDHGA1; NM_018912; 3′UTR; PCDHGA11; NM_032092; 3′UTR; PCDHGA3; NM_018916; 3′UTR; PCDHGA2; NM_018915; 3′UTR; PCDHGB6; NM_018926; 3′UTR; PCDHGA11; NM_018914; 3′UTR; PCDHGC5; NM_018929; 3′UTR; PCDHGA4; NM_018917; 3′UTR; PCDHGB2; NM_018923; 3′UTR; PCDHGA10; NM_018913; 3′UTR; PCDHGC4; NM_018928; 3′UTR; PCDHGB7; NM_018927; 3′UTR; PCDHGA9; NM_018921; 3′UTR; PCDHGC3; NM_032403; 3′UTR; PCDHGB1; NM_018922; 3′UTR; PCDHGA7 NM_018920 3′UTR cg05508296 chr5:1016460 0.24 0.55 NKD2 NM_033120 Body NA NA cg05633070 chr17:933118 0.52 0.78 ABR; NM_021962; Body; chr17:933026- Island ABR; NM_001092; Body; 933236 ABR NM_001159746 Body cg05810177 chr7:134050402 0.49 0.2 NA NA NA NA NA cg05824218 chr17:38499096 0.49 0.7 RARA; NM_001024809; 1stExon; chr17:38497527- S_Shore RARA; NM_001145301; Body; 38498963 RARA; NM_000964; Body; RARA NM_001145302 Body cg05938207 chr6:32489750 0.51 0.29 HLA- NM_002125 Body chr6:32489742- Island DRB5 32490128 cg05949913 chr6:132557557 0.65 0.42 NA NA NA NA NA cg06486129 chr21:45573410 0.14 0.36 NA NA NA chr21:45575451- N_Shelf 45575833 cg06493806 chr18:77278806 0.89 0.57 NFATC1; NM_006162; Body; chr18:77280144- N_Shore NFATC1; NM_172388; Body; 77280414 NFATC1; NM_172389; Body; NFATC1 NM_172387 Body cg06620723 chr1:12404945 0.41 0.68 VPS13D; NM_015378; Body; NA NA VPS13D NM_018156 Body cg06766860 chr12:132863983 0.44 0.66 GALNT9 NM_001122636 Body chr12:132865236- N_Shore 132865506 cg06844165 chr17:3768599 0.84 0.57 CAMKK1; NM_172206; Body; chr17:3769663- N_Shore CAMKK1; NM_032294; Body; 3769874 CAMKK1 NM_172207 3′UTR cg06871529 chr13:32700732 0.8 0.6 FRY NM_023037 Body NA NA cg07030794 chr17:72589110 0.78 0.57 CD300LD; NM_001115152; TSS1500; NA NA C17orf77 NM_152460 3′UTR cg07525313 chr6:116262856 0.57 0.78 FRK NM_002031 3′UTR NA NA cg07857040 chr16:1582219 0.33 0.54 IFT140 NM_014714 Body chr16:1583809- N_Shore 1584641 cg07891658 chr16:87996731 0.27 0.55 BANP; NM_017869; 5′UTR; NA NA BANP NM_079837 5′UTR cg07943832 chr6:155568918 0.74 0.44 TIAM2; NM_012454; Body; NA NA TIAM2 NM_001010927 Body cg08161142 chr13:24273617 0.39 0.65 NA NA NA chr13:24269499- S_Shelf 24270116 cg08233148 chr17:81047721 0.81 0.53 METRNL NM_001004431 Body chr17:81047515- Island 81047965 cg08365687 chr3:52569147 0.33 0.53 NT5DC2; NM_022908; TSS200; chr3:52570475- N_Shore NT5DC2; NM_001134231; TSS1500; 52570842 LOC440957 NM_001124767 TSS1500 cg08797704 chr16:65692605 0.45 0.16 NA NA NA NA NA cg09259081 chr16:84538889 0.32 0.55 KIAA1609 NM_020947 TSS1500 chr16:84538884- Island 84539115 cg09592546 chr17:78652902 0.58 0.88 RPTOR; NM_001163034; Body; NA NA RPTOR NM_020761 Body cg09663736 chr11:131554122 0.75 0.36 NTM NM_001048209 Body NA NA cg09949775 chr19:18902107 0.1 0.33 COMP; NM_000095; 1stExon; chr19:18899037- Island COMP NM_000095 5′UTR 18902284 cg10167378 chr1:228756711 0.85 0.59 NA NA NA chr1:228744110- Island 228784168 cg10270430 chr6:34024362 0.19 0.4 GRM4 NM_000841 Body chr6:34024201- Island 34024457 cg10332003 chr4:185070366 0.66 0.44 ENPP6 NM_153343 Body NA NA cg10590622 chr4:96760945 0.61 0.14 PDHA2 NM_005390 TSS1500 NA NA cg10776061 chr19:12768390 0.54 0.29 MAN2B1 NM_000528 Body chr19:12767749- S_Shore 12767980 cg11035303 chr3:43465503 0.32 0.06 ANO10 NM_018075 Body NA NA cg11317459 chr13:21872234 0.08 0.34 NA NA NA chr13:21872179- Island 21872665 cg11639130 chr12:131303478 0.5 0.72 STX2; NM_001980; Body; chr12:131303093- Island STX2 NM_194356 Body 131303836 cg11728145 chr2:1658190 0.72 0.43 PXDN NM_012293 Body NA NA cg11728747 chr7:29037910 0.29 0.5 CPVL; NM_031311; Body; NA NA CPVL NM_019029 Body cg11791078 chr5:36273196 0.89 0.43 RANBP3L; NM_145000; Body; NA NA RANBP3L NM_001161429 Body cg11986643 chr6:32634316 0.51 0.29 HLA- NM_002123 1stExon chr6:32632158- S_Shore DQB1 32633027 cg12214399 chr4:53210660 0.14 0.55 NA NA NA NA NA cg12293347 chr8:1117672 0.74 0.48 NA NA NA chr8:1113058- S_Shelf 1114073 cg12360123 chr10:79984532 0.56 0.35 NA NA NA NA NA cg12734688 chr1:48308390 0.53 0.32 NA NA NA NA NA cg12743416 chr7:138229989 0.28 0.64 TRIM24; NM_003852; Body; NA NA TRIM24 NM_015905 Body cg12823953 chr3:137893743 0.12 0.34 DBR1; NM_016216; 1stExon; chr3:137893410- Island DBR1 NM_016216 5′UTR 137893808 cg13038618 chr14:77467391 0.37 0.6 NA NA NA NA NA cg13205848 chr12:5675505 0.74 0.52 ANO2 NM_020373 Body NA NA cg13422830 chr1:19985666 0.32 0.67 NA NA NA NA NA cg13506281 chr13:29914200 0.55 0.83 MTUS2 NM_001033602 Body chr13:29913886- Island 29914301 cg13617837 chr6:3724690 0.34 0.54 C6orf145 NM_183373 Body NA NA cg13730219 chr13:21896301 0.54 0.12 NA NA NA chr13:21894085- S_Shore 21894606 cg13749548 chr14:75722495 0.66 0.23 NA NA NA chr14:75725748- N_Shelf 75726029 cg13943068 chr4:1580193 0.11 0.49 NA NA NA chr4:1580050- Island 1580455 cg14173968 chr6:39740405 0.58 0.78 NA NA NA NA NA cg14223671 chr16:857981 0.08 0.29 PRR25 NM_001013638 Body chr16:857341- Island 858025 cg14447606 chr2:72370328 0.11 0.44 CYP26B1 NM_019885 Body chr2:72370296- Island 72370682 cg14456004 chr13:21872349 0.14 0.45 NA NA NA chr13:21872179- Island 21872665 cg14463164 chr9:109715708 0.85 0.54 ZNF462; NM_021224; Body; NA NA MIR548Q NR_031752 Body cg14646613 chr9:110412708 0.78 0.39 NA NA NA NA NA cg14651435 chr7:157209551 0.96 0.68 DNAJB6 NM_058246 3′UTR chr7:157208794- S_Shore 157209008 cg14852082 chr4:1580132 0.25 0.49 NA NA NA chr4:1580050- Island 1580455 cg14895374 chr8:28930481 0.71 0.42 KIF13B NM_015254 Body chr8:28928996- S_Shore 28929718 cg15260248 chr3:189829092 0.64 0.85 LEPREL1; NM_001134418; 5′UTR; NA NA LEPREL1 NM_018192 Body cg15497834 chr20:48998834 0.37 0.61 NA NA NA NA NA cg15690379 chr3:52683739 0.57 0.36 PBRM1; NM_181042; Body; NA NA PBRM1; NM_018313; Body; PBRM1 NM_018165 Body cg15752756 chr6:32634481 0.45 0.17 HLA- NM_002123 TSS200 chr6:32632158- S_Shore DQB1 32633027 cg15878909 chr12:8380286 0.58 0.32 FAM90A1 NM_018088 TSS200 NA NA cg16081854 chr5:308268 0.8 0.07 AHRR; NM_020731; Body; chr5:309705- N_Shore PDCD6 NM_013232 Body 310136 cg16112880 chr1:201123745 0.14 0.67 TMEM9 NM_016456 TSS200 chr1:201123245- Island 201123746 cg16463697 chr2:223886480 0.33 0.73 NA NA NA NA NA cg16508714 chr10:98425110 0.68 0.46 PIK3AP1 NM_152309 Body NA NA cg16540391 chr6:151042035 0.48 0.28 PLEKHG1 NM_001029884 5′UTR NA NA cg16664523 chr5:67586170 0.33 0.53 PIK3R1; NM_181523; Body; chr5:67584213- S_Shore PIK3R1; NM_181504; TSS1500; 67584451 PIK3R1 NM_181524 Body cg17013691 chr5:14380323 0.83 0.62 TRIO NM_007118 Body NA NA cg17171539 chr1:59398690 0.7 0.49 NA NA NA NA NA cg17449954 chr1:40105667 0.15 0.4 HEYL NM_014571 TSS1500 chr1:40105010- Island 40105707 cg17602481 chr13:114890515 0.89 0.59 RASA3 NM_007368 Body NA NA cg17662493 chr22:45806309 0.65 0.88 SMC1B NM_148674 Body chr22:45809191- N_Shelf 45809953 cg17811452 chr20:44007674 0.71 0.45 TP53TG5; NM_014477; TSS1500; NA NA SYS1- NR_003189 Body DBNDD2 cg18004235 chr2:19808330 0.73 0.49 NA NA NA NA NA cg18009021 chr4:95376488 0.75 0.44 PDLIM5; NR_024179; Body; chr4:95372801- S_Shelf PDLIM5; NM_001011515; Body; 95373535 PDLIM5; NM_006457; Body; PDLIM5; NM_001011516; Body; PDLIM5 NM_001011513 Body cg18149745 chr3:197094595 0.77 0.49 NA NA NA NA NA cg18235100 chr4:81124600 0.43 0.65 PRDM8; NM_001099403; Body; chr4:81124468- Island PRDM8 NM_020226 Body 81124845 cg18302225 chr5:55776401 0.23 0.55 NA NA NA chr5:55776604- N_Shore 55777233 cg18332838 chr8:126698738 0.47 0.27 NA NA NA NA NA cg18379295 chr14:52326155 0.29 0.6 GNG2 NM_053064 TSS1500 NA NA cg18438894 chr1:184377525 0.61 0.4 C1orf21 NM_030806 5′UTR NA NA cg18642369 chr13:99651231 0.37 0.64 DOCK9; NM_015296; Body; NA NA DOCK9 NM_001130049 Body cg19141316 chr11:19750209 0.83 0.59 NAV2; NM_182964; Body; NA NA NAV2; NM_001111018; Body; NAV2 NM_145117 Body cg19243721 chr6:166851830 0.37 0.83 RPS6KA2; NM_021135; Body; NA NA RPS6KA2 NM_001006932 Body cg19264571 chr18:10454085 0.26 0.47 APCDD1 NM_153000 TSS1500 chr18:10454082- Island 10454296 cg19300401 chr6:16962712 0.39 0.81 NA NA NA NA NA cg19539986 chr14:35032169 0.9 0.65 SNX6; NM_021249; 3′UTR; NA NA SNX6 NM_152233 3′UTR cg19577074 chr4:152405174 0.32 0.53 FAM160A1 NM_001109977 5′UTR NA NA cg19697575 chr2:172374119 0.57 0.87 NA NA NA chr2:172373817- Island 172374199 cg19717773 chr7:2847554 0.26 0.5 GNA12 NM_007353 Body NA NA cg19799454 chr7:64328759 0.82 0.57 NA NA NA NA NA cg19907305 chr19:18902117 0.12 0.34 COMP NM_000095 TSS200 chr19:18899037- Island 18902284 cg20274462 chr8:95980625 0.71 0.5 NA NA NA NA NA cg20321086 chr8:62052207 0.47 0.25 NA NA NA chr8:62051646- Island 62052431 cg20346503 chr2:128994402 0.46 0.24 NA NA NA chr2:128990509- S_Shelf 128991325 cg20539283 chr2:162932048 0.78 0.47 DPP4 NM_001935 TSS1500 chr2:162930233- S_Shore 162930879 cg20895691 chr2:23641550 0.54 0.26 KLHL29 NM_052920 5′UTR NA NA cg20976286 chr15:28054345 0.59 0.11 OCA2 NM_000275 Body chr15:28050250- S_Shelf 28050789 cg21211688 chr9:136403935 0.81 0.34 ADAMTSL2; NM_014694; Body; chr9:136399367- S_Shelf ADAMTSL2 NM_001145320 Body 136400274 cg21332500 chr7:27233480 0.16 0.37 NA NA NA chr7:27231805- S_Shore 27233097 cg21446981 chr7:37534909 0.49 0.23 NA NA NA NA NA cg21498547 chr8:1651128 0.1 0.62 DLGAP2 NM_004745 3′UTR chr8:1649439- S_Shore 1649759 cg21565914 chr2:162931175 0.72 0.47 DPP4 NM_001935 TSS200 chr2:162930233- S_Shore 162930879 cg21681643 chr2:114039512 0.9 0.66 LOC440839 NR_029399 Body chr2:114034594- S_Shelf 114036041 cg21860675 chr3:71586357 0.6 0.25 FOXP1; NM_032682; 5′UTR; NA NA FOXP1 NM_001012505 5′UTR cg21945639 chr1:200271342 0.39 0.14 NA NA NA chr1:200271276- Island 200271538 cg21964662 chr13:79234715 0.46 0.69 RNF219 NM_024546 TSS1500 chr13:79232822- S_Shore 79233417 cg22031873 chr4:143765657 0.86 0.61 INPP4B; NM_001101669; 5′UTR; chr4:143766940- N_Shore INPP4B NM_003866 5′UTR 143768413 cg22749855 chr17:76353952 0.5 0.29 SOCS3 NM_003955 3′UTR chr17:76354818- N_Shore 76357038 cg23052585 chr10:50328538 0.58 0.82 NA NA NA NA NA cg23159970 chr12:2690385 0.9 0.16 CACNA1C; NM_001129844; Body; NA NA CACNA1C; NM_001129827; Body; CACNA1C; NM_001129839; Body; CACNA1C; NM_001129834; Body; CACNA1C; NM_001129841; Body; CACNA1C; NM_000719; Body; CACNA1C; NM_001129830; Body; CACNA1C; NM_001167625; Body; CACNA1C; NM_001129843; Body; CACNA1C; NM_001167624; Body; CACNA1C; NM_001129835; Body; CACNA1C; NM_001129837; Body; CACNA1C; NM_001167623; Body; CACNA1C; NM_001129840; Body; CACNA1C; NM_199460; Body; CACNA1C; NM_001129833; Body; CACNA1C; NM_001129832; Body; CACNA1C; NM_001129829; Body; CACNA1C; NM_001129846; Body; CACNA1C; NM_001129836; Body; CACNA1C; NM_001129838; Body; CACNA1C; NM_001129831; Body; CACNA1C NM_001129842 Body cg23192683 chr3:194208907 0.67 0.45 NA NA NA chr3:194207385- S_Shore 194208785 cg23677311 chr8:25061108 0.75 0.51 DOCK5 NM_024940 Body NA NA cg23698271 chr10:121346762 0.63 0.89 TIAL1; NM_001033925; Body; NA NA TIAL1 NM_003252 Body cg23763647 chr10:4868690 0.29 0.05 AKR1E2 NM_001040177 Body chr10:4868125- Island 4868949 cg24199384 chr13:24365000 0.57 0.34 MIPEP NM_005932 Body NA NA cg24284539 chr10:12999599 0.75 0.47 CCDC3 NM_031455 Body NA NA cg24451872 chr1:32177995 0.4 0.16 NA NA NA chr1:32180131- N_Shelf 32180487 cg24623760 chr12:123610989 0.57 0.27 NA NA NA NA NA cg25191304 chr1:42097792 0.74 0.49 HIVEP3; NM_024503; 5′UTR; NA NA HIVEP3 NM_001127714 5′UTR cg25491704 chr6:33048879 0.32 0.64 HLA- NM_002121 Body chr6:33048416- S_Shore DPB1 33048814 cg25541928 chr15:51973199 0.71 0.48 SCG3; NM_013243; TSS1500; chr15:51973533- N_Shore SCG3 NM_001165257 TSS1500 51973838 cg25570222 chr2:45804631 0.66 0.91 SRBD1 NM_018079 Body NA NA cg25614253 chr8:143561205 0.33 0.12 BAI1 NM_001702 Body chr8:143558487- S_Shelf 143558824 cg25638870 chr11:89224717 0.18 0.42 NOX4; NM_001143837; 5′UTR; chr11:89224416- Island NOX4; NR_026571; TSS200; 89224718 NOX4; NM_016931; TSS200; NOX4 NM_001143836 TSS200 cg25909532 chr7:158821175 0.57 0.33 VIPR2 NM_003382 3′UTR chr7:158823178- N_Shelf 158824316 cg25929399 chr17:39597601 0.39 0.16 KRT38 NM_006771 TSS200 NA NA cg26365090 chr20:42574362 0.87 0.47 TOX2; NM_001098796; 5′UTR; NA NA TOX2; NM_001098797; Body; TOX2; NM_001098798; TSS200; TOX2 NM_032883 5′UTR cg26646659 chr5:55776364 0.31 0.52 NA NA NA chr5:55776604- N_Shore 55777233 cg26690407 chr5:5887642 0.44 0.17 NA NA NA chr5:5887062- S_Shore 5887528 cg26853458 chr17:9805074 0.24 0.49 RCVRN NM_002903 Body chr17:9808067- N_Shelf 9808339 cg26932889 chr1:54135470 0.28 0.49 GLIS1 NM_147193 5′UTR NA NA cg27010076 chr5:112586110 0.72 0.38 MCC; NM_002387; Body; NA NA MCC NM_001085377 Body cg27031754 chr5:54185940 0.47 0.26 NA NA NA NA NA cg27065717 chr16:85608934 0.46 0.67 NA NA NA NA NA cg27286614 chr7:2050401 0.46 0.84 MAD1L1; NM_003550; Body; chr7:2054060- N_Shelf MAD1L1; NM_001013837; Body; 2054386 MAD1L1 NM_001013836 Body cg27333018 chr19:2897514 0.69 0.47 NA NA NA chr19:2900329- N_Shelf 2901203 Hidden DMR Markov DHS (differentially Model Regulatory_(—) Regulatory_(—) (DNAse I methylated (HMM)_(—) Feature_(—) Feature_(—) hypersensitive Name Phantom region) Enhancer Island Name Group site) cg00156230 NA NA TRUE NA NA NA NA cg00264799 NA NA TRUE NA NA NA NA cg00290607 NA NA TRUE 11: 67140114- 11: 67382913- Promoter_(—) NA 67140331 67383979 Associated cg00481216 NA NA TRUE NA NA NA NA cg00518941 NA NA TRUE NA NA NA NA cg00575645 NA NA TRUE NA NA NA NA cg00806704 NA NA NA NA NA NA NA cg00945409 low- NA TRUE NA NA NA TRUE CpG: 80407669- 80407738 cg01230320 NA NA NA 2: 19413297- 2: 19549091- Promoter_(—) TRUE 19414688 19550340 Associated cg01395541 NA NA NA NA NA NA NA cg01468567 NA NA TRUE NA 19: 49843436- Unclassified NA 49843948 cg01470456 NA NA TRUE NA NA NA NA cg01549977 NA NA TRUE NA NA NA TRUE cg01632562 NA NA NA NA NA NA NA cg01694488 NA NA NA 4: 1550144- NA NA NA 1550508 cg01790920 NA NA NA 5: 3647396- NA NA NA 3649997 cg02100397 NA NA NA 19: 596804- NA NA NA 598501 cg02138953 NA NA TRUE NA NA NA NA cg02159489 NA NA NA 17: 77074036- NA NA NA 77074159 cg02197634 NA NA TRUE NA NA NA NA cg02368820 NA NA NA 1: 3041863- NA NA NA 3042751 cg02395396 NA NA TRUE NA NA NA TRUE cg02527375 NA NA TRUE NA NA NA NA cg02532518 NA NA NA 16: 3149986- 16: 3209865- Unclassified_(—) NA 3150476 3210112 Cell_type_(—) specific cg02799905 NA NA TRUE NA NA NA NA cg02890259 NA NA TRUE NA NA NA TRUE cg03079497 NA NA NA 17: 1336914- 17: 1390020- Unclassified TRUE 1337650 1390909 cg03088219 NA NA NA NA NA NA NA cg03119829 NA DMR NA NA 1: 64170893- Unclassified_(—) NA 64170969 Cell_type_(—) specific cg03217995 NA DMR NA 7: 27169827- NA NA NA 27171776 cg03301058 NA DMR TRUE NA 6: 90007623- Unclassified TRUE 90008148 cg03487027 NA NA NA 10: 76828340- 10: 77158878- Unclassified_(—) NA 76830392 77159061 Cell_type_(—) specific cg03653601 NA NA TRUE NA 16: 16155825- Unclassified_(—) NA 16156219 Cell_type_(—) specific cg03814093 NA NA NA 4: 154629395- 4: 154409197- Promoter_(—) NA 154629457 154410467 Associated cg03859028 NA NA TRUE NA 15: 99948962- Unclassified TRUE 99949571 cg04478875 NA NA TRUE NA NA NA NA cg04500819 NA NA TRUE NA NA NA NA cg04506342 NA NA TRUE NA NA NA NA cg04887066 NA NA TRUE NA NA NA NA cg04888234 NA NA NA NA NA NA NA cg04894537 NA NA TRUE NA NA NA TRUE cg04998634 NA NA NA 19: 1807744- 19: 1856867- Promoter_(—) NA 1808443 1857574 Associated cg05210689 NA NA TRUE NA NA NA TRUE cg05227215 NA NA TRUE NA NA NA NA cg05276972 NA NA TRUE NA NA NA TRUE cg05279330 NA NA TRUE NA NA NA NA cg05422883 NA NA TRUE NA NA NA NA cg05424060 NA NA NA NA NA NA NA cg05494467 NA CDMR NA 5: 140872467- 5: 140891341- Promoter_(—) TRUE 140872619 140892957 Associated cg05508296 NA NA NA 5: 1069176- NA NA TRUE 1069496 cg05633070 NA NA TRUE 17: 879777- NA NA NA 879966 cg05810177 NA NA TRUE NA NA NA NA cg05824218 NA NA NA 17: 35751130- NA NA NA 35752623 cg05938207 NA NA NA 6: 32597721- NA NA NA 32598106 cg05949913 NA NA TRUE NA NA NA NA cg06486129 NA NA NA NA 21: 45573348- Unclassified TRUE 45574201 cg06493806 NA NA NA 18: 75379223- NA NA NA 75379816 cg06620723 low- NA NA NA 1: 12404910- Unclassified_(—) NA CpG: 12405164 Cell_type_(—) 12327488- specific 12327568 cg06766860 NA NA NA 12: 131374053- NA NA NA 131374345 cg06844165 NA NA NA NA NA NA TRUE cg06871529 NA NA TRUE NA NA NA NA cg07030794 NA NA NA NA NA NA NA cg07525313 NA NA NA NA NA NA NA cg07857040 NA NA NA 16: 1522055- 16: 1582161- Unclassified NA 1522410 1582500 cg07891658 NA NA NA 16: 86554232- 16: 87996629- Promoter_(—) NA 86554374 87997495 Associated_(—) Cell_type_(—) specific cg07943832 NA NA TRUE NA 6: 155568903- Unclassified TRUE 155570209 cg08161142 NA NA NA NA NA NA NA cg08233148 NA NA NA 17: 78640438- NA NA NA 78641204 cg08365687 NA NA NA NA 3: 52569028- Unclassified_(—) NA 52569356 Cell_type_(—) specific cg08797704 NA NA TRUE NA NA NA TRUE cg09259081 NA NA NA 16: 83096386- 16: 84537881- Promoter_(—) NA 83096616 84539086 Associated cg09592546 NA NA TRUE NA 17: 78652494- Unclassified_(—) NA 78653143 Cell_type_(—) specific cg09663736 NA NA TRUE NA NA NA NA cg09949775 NA NA NA 19: 18760038- NA NA NA 18763274 cg10167378 NA NA NA 1: 226821657- NA NA NA 226823404 cg10270430 NA NA TRUE 6: 34132180- NA NA TRUE 34132458 cg10332003 NA NA TRUE NA 4: 185070345- Unclassified_(—) NA 185070459 Cell_type_(—) specific cg10590622 NA NA NA NA NA NA NA cg10776061 NA NA TRUE 19: 12628423- NA NA TRUE 12629391 cg11035303 NA NA TRUE NA NA NA NA cg11317459 NA NA NA 13: 20770180- 13: 21870878- Promoter_(—) NA 20770868 21873080 Associated cg11639130 NA NA TRUE 12: 129869077- 12: 131302803- Promoter_(—) TRUE 129869434 131304615 Associated cg11728145 NA NA NA 2: 1637173- NA NA TRUE 1637241 cg11728747 NA NA TRUE NA NA NA NA cg11791078 NA NA NA NA NA NA NA cg11986643 NA NA NA NA 6: 32634123- Unclassified TRUE 32634701 cg12214399 NA NA TRUE NA NA NA NA cg12293347 NA RDMR NA NA NA NA NA cg12360123 NA NA TRUE NA NA NA NA cg12734688 NA NA TRUE NA NA NA NA cg12743416 NA NA NA NA NA NA NA cg12823953 low- NA NA 3: 139376110- 3: 137892888- Promoter_(—) NA CpG: 139376498 137894011 Associated 139376377- 139376484 cg13038618 NA NA NA 14: 76536767- NA NA NA 76537145 cg13205848 NA NA TRUE NA 12: 5674812- Unclassified_(—) NA 5675561 Cell_type_(—) specific cg13422830 NA NA TRUE NA NA NA NA cg13506281 NA NA NA 13: 28811887- NA NA NA 28812268 cg13617837 NA NA TRUE 6: 3669402- NA NA TRUE 3669690 cg13730219 NA NA NA NA NA NA NA cg13749548 NA NA NA NA NA NA NA cg13943068 NA NA NA 4: 1550144- NA NA NA 1550508 cg14173968 NA NA TRUE NA NA NA NA cg14223671 NA NA NA 16: 797343- 16: 857066- Promoter_(—) NA 798706 858592 Associated cg14447606 NA NA TRUE 2: 72223562- NA NA NA 72226253 cg14456004 NA NA NA 13: 20770180- 13: 21870878- Promoter_(—) NA 20770868 21873080 Associated cg14463164 NA NA TRUE NA NA NA NA cg14646613 NA NA TRUE NA NA NA NA cg14651435 NA NA NA 7: 156902292- NA NA NA 156902330 cg14852082 NA NA NA 4: 1550144- NA NA NA 1550508 cg14895374 NA NA NA NA NA NA TRUE cg15260248 NA NA TRUE NA NA NA NA cg15497834 NA DMR NA NA 20: 48998337- Unclassified_(—) NA 48999129 Cell_type_(—) specific cg15690379 NA NA TRUE NA NA NA NA cg15752756 low- NA NA NA 6: 32634123- Unclassified TRUE CpG: 32634701 32742410- 32742475 cg15878909 NA NA NA NA NA NA NA cg16081854 NA NA NA 5: 361235- NA NA NA 361628 cg16112880 NA NA NA 1: 199389889- 1: 201122822- Promoter_(—) NA 199390390 201124261 Associated cg16463697 NA NA TRUE NA NA NA NA cg16508714 NA NA TRUE NA NA NA NA cg16540391 NA NA TRUE NA 6: 151041999- Unclassified_(—) NA 151042157 Cell_type_(—) specific cg16664523 NA NA TRUE NA NA NA NA cg17013691 NA NA TRUE 5: 14433305- NA NA NA 14433455 cg17171539 NA NA TRUE NA NA NA TRUE cg17449954 high- NA NA 1: 39877598- 1: 40104740- Unclassified_(—) TRUE CpG: 39878275 40105984 Cell_type_(—) 39877885- specific 39878287 cg17602481 NA NA NA 13: 113908518- 13: 114890255- Unclassified_(—) TRUE 113908715 114890920 Cell_type_(—) specific cg17662493 NA NA NA NA NA NA NA cg17811452 NA NA NA NA NA NA NA cg18004235 NA NA TRUE NA NA NA NA cg18009021 low- NA NA NA NA NA NA CpG: 95595502- 95595528 cg18149745 NA NA TRUE NA NA NA TRUE cg18235100 NA NA NA 4: 81342012- 4: 81124393- Promoter_(—) TRUE 81343857 81124763 Associated cg18302225 NA CDMR TRUE 5: 55812102- NA NA TRUE 55812159 cg18332838 NA NA TRUE NA NA NA NA cg18379295 NA NA NA NA NA NA NA cg18438894 NA NA TRUE NA NA NA NA cg18642369 NA NA TRUE NA 13: 99651046- Unclassified_(—) NA 99651440 Cell_type_(—) specific cg19141316 NA NA TRUE NA NA NA NA cg19243721 NA NA NA 6: 166771808- NA NA NA 166771895 cg19264571 NA RDMR NA 18: 10443763- NA NA TRUE 10445606 cg19300401 NA NA TRUE NA NA NA NA cg19539986 NA NA NA NA NA NA NA cg19577074 NA NA TRUE NA NA NA NA cg19697575 NA NA NA 2: 172082064- NA NA NA 172082421 cg19717773 NA NA NA 7: 2814039- NA NA NA 2814102 cg19799454 NA NA NA 7: 63966088- NA NA NA 63966326 cg19907305 high- NA NA 19: 18760038- NA NA NA CpG: 18763274 18763111- 18763118 cg20274462 NA NA TRUE NA NA NA NA cg20321086 NA DMR TRUE 8: 62214562- 8: 62051046- Unclassified TRUE 62215149 62052442 cg20346503 NA NA TRUE NA NA NA TRUE cg20539283 NA NA NA NA NA NA NA cg20895691 NA NA TRUE NA NA NA TRUE cg20976286 NA CDMR NA NA NA NA NA cg21211688 NA NA NA NA NA NA NA cg21332500 NA RDMR NA 7: 27199936- NA NA NA 27200029 cg21446981 NA NA TRUE NA NA NA NA cg21498547 NA NA NA 8: 1638463- NA NA NA 1638636 cg21565914 NA NA TRUE NA 2: 162929683- Promoter_(—) TRUE 162931506 Associated cg21681643 NA NA NA NA NA NA NA cg21860675 NA NA TRUE NA NA NA TRUE cg21945639 NA NA TRUE 1: 198537912- 1: 200270611- Promoter_(—) NA 198538577 200272226 Associated_(—) Cell_type_(—) specific cg21964662 NA NA NA NA NA NA NA cg22031873 NA NA NA NA NA NA NA cg22749855 NA NA NA NA 17: 76353631- Promoter_(—) NA 76354680 Associated cg23052585 NA NA NA NA NA NA NA cg23159970 NA NA TRUE NA NA NA NA cg23192683 NA NA TRUE NA NA NA NA cg23677311 NA NA TRUE NA NA NA NA cg23698271 NA NA TRUE NA NA NA NA cg23763647 NA DMR NA 10: 4858068- 10: 4867931- Unclassified_(—) TRUE 4858934 4869103 Cell_type_(—) specific cg24199384 NA NA TRUE NA NA NA NA cg24284539 NA NA NA 10: 13039521- NA NA NA 13039676 cg24451872 NA NA NA NA NA NA NA cg24623760 NA NA TRUE NA 12: 123609968- Unclassified_(—) TRUE 123611863 Cell_type_(—) specific cg25191304 NA NA TRUE NA NA NA NA cg25491704 NA NA TRUE NA NA NA NA cg25541928 NA NA NA NA NA NA NA cg25570222 NA NA TRUE NA NA NA NA cg25614253 NA NA NA 8: 143557823- 8: 143561069- Unclassified_(—) NA 143558304 143561351 Cell_type_(—) specific cg25638870 NA NA NA 11: 88864029- NA NA NA 88864366 cg25909532 NA NA NA NA NA NA TRUE cg25929399 NA NA NA NA NA NA NA cg26365090 NA NA NA NA NA NA NA cg26646659 NA CDMR TRUE 5: 55812102- NA NA TRUE 55812159 cg26690407 NA NA NA NA NA NA NA cg26853458 NA NA NA NA NA NA NA cg26932889 NA NA TRUE NA NA NA NA cg27010076 NA NA TRUE NA NA NA NA cg27031754 NA NA TRUE NA NA NA TRUE cg27065717 NA NA NA 16: 84166244- NA NA NA 84166479 cg27286614 NA NA NA 7: 2016696- NA NA NA 2017035 cg27333018 NA NA NA NA NA NA NA

In some embodiments, the fibroblast, e.g., derived from a wound, such as a diabetic ulcer (e.g., diabetic foot ulcer), comprises an elevated level of a protein marker, e.g., fibronectin. For example, the level of the protein marker, e.g., fibronectin, is increased by at least 2-fold (e.g., at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more). In other example, the fibroblast, e.g., derived from a wound, such as a diabetic ulcer (e.g., diabetic foot ulcer), comprises decreased level of a protein marker, e.g., smooth muscle actin or plasminogen activator inhibitor-1 (PAI-1, also called Serpin E1). For example, the level of the protein marker, e.g., smooth muscle actin or PAI-1, is decreased by at least 2-fold (e.g., at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more).

For example, the amino acid sequence of human fibronectin is provided by GenBank Accession No. P02751.4 and is shown below:

(SEQ ID NO: 17)    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq   61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi  121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvol gngkgewtck  181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy  241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp  301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc  361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc  421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri  481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm  541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq  601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip  661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp  721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl  781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdttvd qvddtsivvr wsrpqapitg  841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg  901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt  961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp 1021 raqitgyrlt vgltrrgqpr qynvgpsysk yplrnlqpas eytvslvaik gnqespkatg 1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv 1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt 1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis 1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd 1321 navvltnllp gteyvvsyss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia 1381 pratitgyri rhhpehfsgr predrvphsr nsitltnitp gteyvvsiva lngreespll 1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk 1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvqdnsisv 1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge 1621 sqplvqtavt nidrpkglaf tdvdvdsiki awespqgqvs ryrvtysspe dgihelfpap 1681 dgeedtaelq glrpgseytv svvalhddme sqpligtqst aipaptdlkf tqvtptslsa 1741 qwtppnvqlt gyrvrvtpke ktgpmkeinl apdsssvvvs glmvatkyev svyalkdtlt 1801 srpaqgvvtt lenvspprra rvtdatetti tiswrtktet itgfqvdavp angqtpiqrt 1861 ikpdvrsyti tglqpgtdyk iylytlndna rsspvvidas taidapsnlr flattpnsll 1921 vswqpprari tgyiikyekp gspprevvpr prpgvteati tglepgteyt iyvialknnq 1981 ksepligrkk tdelpqlvtl phpnlhgpei ldvpstvqkt pfvthpgydt gngiqlpgts 2041 gqqpsvgqqm ifeehgfrrt tppttatpir hrprpyppnv geeiqighip redvdyhlyp 2101 hgpglnpnas tgqealsqtt iswapfqdts eyiischpvg tdeeplqfry pgtstsatlt 2161 gltrgatynv ivealkdqqr hkvreevvtv gnsvneglnq ptddscfdpy tvshyavgde 2221 wermsesgfk llcgclgfgs ghfrcdssrw chdngvnyki gekwdrggen gqmmsctclg 2281 ngkgefkcdp heatcyddgk tyhvgeqwqk eylgaicsct cfggqrgwrc dncrrpggep 2341 spegttgqsy nqysgryhqr tntnvncpie cfmpldvqad redsre 

The mRNA sequence encoding human Fibronectin is provided by GenBank Accesion No. _212482.1, and the sequence is shown below. Start and stop codons are in bold and underlined.

(SEQ ID NO: 18)    1 gcccgcgccg gctgtgctgc acagggggag gagagggaac cccaggcgcg agcgggaaga   61 ggggacctgc agccacaact tctctggtcc tctgcatccc ttctgtccct ccacccgtcc  121 ccttccccac cctctggccc ccaccttctt ggaggcgaca acccccggga ggcattagaa  181 gggatttttc ccgcaggttg cgaagggaag caaacttggt ggcaacttgc ctcccggtgc  241 gggcgtctct cccccaccgt ctcaac atg c ttaggggtcc ggggcccggg ctgctgctgc  301 tggccgtcca gtgcctgggg acagcggtgc cctccacggg agcctcgaag agcaagaggc  361 aggctcagca aatggttcag ccccagtccc cggtggctgt cagtcaaagc aagcccggtt  421 gttatgacaa tggaaaacac tatcagataa atcaacagtg ggagcggacc tacctaggca  481 atgcgttggt ttgtacttgt tatggaggaa gccgaggttt taactgcgag agtaaacctg  541 aagctgaaga gacttgcttt gacaagtaca ctgggaacac ttaccgagtg ggtgacactt  601 atgagcgtcc taaagactcc atgatctggg actgtacctg catcggggct gggcgaggga  661 gaataagctg taccatcgca aaccgctgcc atgaaggggg tcagtcctac aagattggtg  721 acacctggag gagaccacat gagactggtg gttacatgtt agagtgtgtg tgtcttggta  781 atggaaaagg agaatggacc tgcaagccca tagctgagaa gtgttttgat catgctgctg  841 ggacttccta tgtggtcgga gaaacgtggg agaagcccta ccaaggctgg atgatggtag  901 attgtacttg cctgggagaa ggcagcggac gcatcacttg cacttctaga aatagatgca  961 acgatcagga cacaaggaca tcctatagaa ttggagacac ctggagcaag aaggataatc 1021 gaggaaacct gctccagtgc atctgcacag gcaacggccg aggagagtgg aagtgtgaga 1081 ggcacacctc tgtgcagacc acatcgagcg gatctggccc cttcaccgat gttcgtgcag 1141 ctgtttacca accgcagcct cacccccagc ctcctcccta tggccactgt gtcacagaca 1201 gtggtgtggt ctactctgtg gggatgcagt ggctgaagac acaaggaaat aagcaaatgc 1261 tttgcacgtg cctgggcaac ggagtcagct gccaagagac agctgtaacc cagacttacg 1321 gtggcaactc aaatggagag ccatgtgtct taccattcac ctacaatggc aggacgttct 1381 actcctgcac cacagaaggg cgacaggacg gacatctttg gtgcagcaca acttcgaatt 1441 atgagcagga ccagaaatac tctttctgca cagaccacac tgttttggtt cagactcgag 1501 gaggaaattc caatggtgcc ttgtgccact tccccttcct atacaacaac cacaattaca 1561 ctgattgcac ttctgagggc agaagagaca acatgaagtg gtgtgggacc acacagaact 1621 atgatgccga ccagaagttt gggttctgcc ccatggctgc ccacgaggaa atctgcacaa 1681 ccaatgaagg ggtcatgtac cgcattggag atcagtggga taagcagcat gacatgggtc 1741 acatgatgag gtgcacgtgt gttgggaatg gtcgtgggga atggacatgc attgcctact 1801 cgcagcttcg agatcagtgc attgttgatg acatcactta caatgtgaac gacacattcc 1861 acaagcgtca tgaagagggg cacatgctga actgtacatg cttcggtcag ggtcggggca 1921 ggtggaagtg tgatcccgtc gaccaatgcc aggattcaga gactgggacg ttttatcaaa 1981 ttggagattc atgggagaag tatgtgcatg gtgtcagata ccagtgctac tgctatggcc 2041 gtggcattgg ggagtggcat tgccaacctt tacagaccta tccaagctca agtggtcctg 2101 tcgaagtatt tatcactgag actccgagtc agcccaactc ccaccccatc cagtggaatg 2161 caccacagcc atctcacatt tccaagtaca ttctcaggtg gagacctaaa aattctgtag 2221 gccgttggaa ggaagctacc ataccaggcc acttaaactc ctacaccatc aaaggcctga 2281 agcctggtgt ggtatacgag ggccagctca tcagcatcca gcagtacggc caccaagaag 2341 tgactcgctt tgacttcacc accaccagca ccagcacacc tgtgaccagc aacaccgtga 2401 caggagagac gactcccttt tctcctcttg tggccacttc tgaatctgtg accgaaatca 2461 cagccagtag ctttgtggtc tcctgggtct cagcttccga caccgtgtcg ggattccggg 2521 tggaatatga gctgagtgag gagggagatg agccacagta cctggatctt ccaagcacag 2581 ccacttctgt gaacatccct gacctgcttc ctggccgaaa atacattgta aatgtctatc 2641 agatatctga ggatggggag cagagtttga tcctgtctac ttcacaaaca acagcgcctg 2701 atgcccctcc tgacccgact gtggaccaag ttgatgacac ctcaattgtt gttcgctgga 2761 gcagacccca ggctcccatc acagggtaca gaatagtcta ttcgccatca gtagaaggta 2821 gcagcacaga actcaacctt cctgaaactg caaactccgt caccctcagt gacttgcaac 2881 ctggtgttca gtataacatc actatctatg ctgtggaaga aaatcaagaa agtacacctg 2941 ttgtcattca acaagaaacc actggcaccc cacgctcaga tacagtgccc tctcccaggg 3001 acctgcagtt tgtggaagtg acagacgtga aggtcaccat catgtggaca ccgcctgaga 3061 gtgcagtgac cggctaccgt gtggatgtga tccccgtcaa cctgcctggc gagcacgggc 3121 agaggctgcc catcagcagg aacacctttg cagaagtcac cgggctgtcc cctggggtca 3181 cctattactt caaagtcttt gcagtgagcc atgggaggga gagcaagcct ctgactgctc 3241 aacagacaac caaactggat gctcccacta acctccagtt tgtcaatgaa actgattcta 3301 ctgtcctggt gagatggact ccacctcggg cccagataac aggataccga ctgaccgtgg 3361 gccttacccg aagaggacag cccaggcagt acaatgtggg tccctctgtc tccaagtacc 3421 cactgaggaa tctgcagcct gcatctgagt acaccgtatc cctcgtggcc ataaagggca 3481 accaagagag ccccaaagcc actggagtct ttaccacact gcagcctggg agctctattc 3541 caccttacaa caccgaggtg actgagacca ccattgtgat cacatggacg cctgctccaa 3601 gaattggttt taagctgggt gtacgaccaa gccagggagg agaggcacca cgagaagtga 3661 cttcagactc aggaagcatc gttgtgtccg gcttgactcc aggagtagaa tacgtctaca 3721 ccatccaagt cctgagagat ggacaggaaa gagatgcgcc aattgtaaac aaagtggtga 3781 caccattgtc tccaccaaca aacttgcatc tggaggcaaa ccctgacact ggagtgctca 3841 cagtctcctg ggagaggagc accaccccag acattactgg ttatagaatt accacaaccc 3901 ctacaaacgg ccagcaggga aattctttgg aagaagtggt ccatgctgat cagagctcct 3961 gcacttttga taacctgagt cccggcctgg agtacaatgt cagtgtttac actgtcaagg 4021 atgacaagga aagtgtccct atctctgata ccatcatccc agaggtgccc caactcactg 4081 acctaagctt tgttgatata accgattcaa gcatcggcct gaggtggacc ccgctaaact 4141 cttccaccat tattgggtac cgcatcacag tagttgcggc aggagaaggt atccctattt 4201 ttgaagattt tgtggactcc tcagtaggat actacacagt cacagggctg gagccgggca 4261 ttgactatga tatcagcgtt atcactctca ttaatggcgg cgagagtgcc cctactacac 4321 tgacacaaca aacggctgtt cctcctccca ctgacctgcg attcaccaac attggtccag 4381 acaccatgcg tgtcacctgg gctccacccc catccattga tttaaccaac ttcctggtgc 4441 gttactcacc tgtgaaaaat gaggaagatg ttgcagagtt gtcaatttct ccttcagaca 4501 atgcagtggt cttaacaaat ctcctgcctg gtacagaata tgtagtgagt gtctccagtg 4561 tctacgaaca acatgagagc acacctctta gaggaagaca gaaaacaggt cttgattccc 4621 caactggcat tgacttttct gatattactg ccaactcttt tactgtgcac tggattgctc 4681 ctcgagccac catcactggc tacaggatcc gccatcatcc cgagcacttc agtgggagac 4741 ctcgagaaga tcgggtgccc cactctcgga attccatcac cctcaccaac ctcactccag 4801 gcacagagta tgtggtcagc atcgttgctc ttaatggcag agaggaaagt cccttattga 4861 ttggccaaca atcaacagtt tctgatgttc cgagggacct ggaagttgtt gctgcgaccc 4921 ccaccagcct actgatcagc tgggatgctc ctgctgtcac agtgagatat tacaggatca 4981 cttacggaga gacaggagga aatagccctg tccaggagtt cactgtgcct gggagcaagt 5041 ctacagctac catcagcggc cttaaacctg gagttgatta taccatcact gtgtatgctg 5101 tcactggccg tggagacagc cccgcaagca gcaagccaat ttccattaat taccgaacag 5161 aaattgacaa accatcccag atgcaagtga ccgatgttca ggacaacagc attagtgtca 5221 agtggctgcc ttcaagttcc cctgttactg gttacagagt aaccaccact cccaaaaatg 5281 gaccaggacc aacaaaaact aaaactgcag gtccagatca aacagaaatg actattgaag 5341 gcttgcagcc cacagtggag tatgtggtta gtgtctatgc tcagaatcca agcggagaga 5401 gtcagcctct ggttcagact gcagtaacca acattgatcg ccctaaagga ctggcattca 5461 ctgatgtgga tgtcgattcc atcaaaattg cttgggaaag cccacagggg caagtttcca 5521 ggtacagggt gacctactcg agccctgagg atggaatcca tgagctattc cctgcacctg 5581 atggtgaaga agacactgca gagctgcaag gcctcagacc gggttctgag tacacagtca 5641 gtgtggttgc cttgcacgat gatatggaga gccagcccct gattggaacc cagtccacag 5701 ctattcctgc accaactgac ctgaagttca ctcaggtcac acccacaagc ctgagcgccc 5761 agtggacacc acccaatgtt cagctcactg gatatcgagt gcgggtgacc cccaaggaga 5821 agaccggacc aatgaaagaa atcaaccttg ctcctgacag ctcatccgtg gttgtatcag 5881 gacttatggt ggccaccaaa tatgaagtga gtgtctatgc tcttaaggac actttgacaa 5941 gcagaccagc tcagggagtt gtcaccactc tggagaatgt cagcccacca agaagggctc 6001 gtgtgacaga tgctactgag accaccatca ccattagctg gagaaccaag actgagacga 6061 tcactggctt ccaagttgat gccgttccag ccaatggcca gactccaatc cagagaacca 6121 tcaagccaga tgtcagaagc tacaccatca caggtttaca accaggcact gactacaaga 6181 tctacctgta caccttgaat gacaatgctc ggagctcccc tgtggtcatc gacgcctcca 6241 ctgccattga tgcaccatcc aacctgcgtt tcctggccac cacacccaat tccttgctgg 6301 tatcatggca gccgccacgt gccaggatta ccggctacat catcaagtat gagaagcctg 6361 ggtctcctcc cagagaagtg gtccctcggc cccgccctgg tgtcacagag gctactatta 6421 ctggcctgga accgggaacc gaatatacaa tttatgtcat tgccctgaag aataatcaga 6481 agagcgagcc cctgattgga aggaaaaaga cagacgagct tccccaactg gtaacccttc 6541 cacaccccaa tcttcatgga ccagagatct tggatgttcc ttccacagtt caaaagaccc 6601 ctttcgtcac ccaccctggg tatgacactg gaaatggtat tcagcttcct ggcacttctg 6661 gtcagcaacc cagtgttggg caacaaatga tctttgagga acatggtttt aggcggacca 6721 caccgcccac aacggccacc cccataaggc ataggccaag accatacccg ccgaatgtag 6781 gtgaggaaat ccaaattggt cacatcccca gggaagatgt agactatcac ctgtacccac 6841 acggtccggg actcaatcca aatgcctcta caggacaaga agctctctct cagacaacca 6901 tctcatgggc cccattccag gacacttctg agtacatcat ttcatgtcat cctgttggca 6961 ctgatgaaga acccttacag ttcagggttc ctggaacttc taccagtgcc actctgacag 7021 gcctcaccag aggtgccacc tacaacatca tagtggaggc actgaaagac cagcagaggc 7081 ataaggttcg ggaagaggtt gttaccgtgg gcaactctgt caacgaaggc ttgaaccaac 7141 ctacggatga ctcgtgcttt gacccctaca cagtttccca ttatgccgtt ggagatgagt 7201 gggaacgaat gtctgaatca ggctttaaac tgttgtgcca gtgcttaggc tttggaagtg 7261 gtcatttcag atgtgattca tctagatggt gccatgacaa tggtgtgaac tacaagattg 7321 gagagaagtg ggaccgtcag ggagaaaatg gccagatgat gagctgcaca tgtcttggga 7381 acggaaaagg agaattcaag tgtgaccctc atgaggcaac gtgttatgat gatgggaaga 7441 cataccacgt aggagaacag tggcagaagg aatatctcgg tgccatttgc tcctgcacat 7501 gctttggagg ccagcggggc tggcgctgtg acaactgccg cagacctggg ggtgaaccca 7561 gtcccgaagg cactactggc cagtcctaca accagtattc tcagagatac catcagagaa 7621 caaacactaa tgttaattgc ccaattgagt gcttcatgcc tttagatgta caggctgaca 7681 gagaagattc ccgagagtaa atcatctttc caatccagag gaacaagcat gtctctctgc 7741 caagatccat ctaaactgga gtgatgttag cagacccagc ttagagttct tctttctttc 7801 ttaagccctt tgctctggag gaagttctcc agcttcagct caactcacag cttctccaag 7861 catcaccctg ggagtttcct gagggttttc tcataaatga gggctgcaca ttgcctgttc 7921 tgcttcgaag tattcaatac cgctcagtat tttaaatgaa gtgattctaa gatttggttt 7981 gggatcaata ggaaagcata tgcagccaac caagatgcaa atgttttgaa atgatatgac 8041 caaaatttta agtaggaaag tcacccaaac acttctgctt tcacttaagt gtctggcccg 8101 caatactgta ggaacaagca tgatcttgtt actgtgatat tttaaatatc cacagtactc 8161 actttttcca aatgatccta gtaattgcct agaaatatct ttctcttacc tgttatttat 8221 caatttttcc cagtattttt atacggaaaa aattgtattg aaaacactta gtatgcagtt 8281 gataagagga atttggtata attatggtgg gtgattattt tttatactgt atgtgccaaa 8341 gctttactac tgtggaaaga caactgtttt aataaaagat ttacattcca caacttgaag 8401 ttcatctatt tgatataaga caccttcggg ggaaataatt cctgtgaata ttctttttca 8461 attcagcaaa catttgaaaa tctatgatgt gcaagtctaa ttgttgattt cagtacaaga 8521 ttttctaaat cagttgctac aaaaactgat tggtttttgt cacttcatct cttcactaat 8581 ggagatagct ttacactttc tgctttaata gatttaagtg gaccccaata tttattaaaa 8641 ttgctagttt accgttcaga agtataatag aaataatctt tagttgctct tttctaacca 8701 ttgtaattct tcccttcttc cctccacctt tccttcattg aataaacctc tgttcaaaga 8761 gattgcctgc aagggaaata aaaatgacta agatattaaa aaaaaaaaaa aaaaa

The amino acid sequence of human smooth muscle actin is provided by GenBank

Accession No. AAH94877.1 and is shown below.

(SEQ ID NO: 19)   1 mceeettalv cdngsglcka gfagddapra vfpsivgrpr hqgvmvgmgq kdsyvgdeaq  61 skrgiltlky piehgiitnw ddmekiwhhs fynelrvape ehptllteap lnpkanrekm 121 tqimfetfnv pamyvaiqav lslyasgrtt givldsgdgv thnvpiyegy alphaimrld 181 lagrdltdyl mkiltergys fvttaereiv rdikeklcyv aldfenemat aassssleks 241 yelpdgqvit ignerfrcpe tlfqpsfigm esagihetty nsimkcdidi rkdlyannvl 301 sggttmypgi adrmqkeita lapstmkiki iapperkysv wiggsilasl stfqqmwisk 361 peydeagpsi vhrkcf 

The mRNA sequence encoding human smooth muscle actin is provided by GenBank Accession No. BC094877.1 and is shown below. The start and stop codons are in bold and underlined.

(SEQ ID NO: 20)    1 aggtttctta aaaaaaacac acagagaaat attgtgctcc agcccccagc tcattccacc   61 gctcccacc a tg tgtgaaga agagaccacc gcccttgtgt gtgacaatgg ctctggcctg  121 tgcaaggcag gctttgcagg agatgatgcc cccagggctg tcttcccctc cattgtgggc  181 cgccctagac atcagggtgt gatggtggga atgggccaga aagacagcta tgtgggggac  241 gaggctcaga gcaagcgtgg gatcctaact ctcaagtacc ctattgaaca tggcatcatc  301 accaactggg atgacatgga gaagatctgg caccactcct tctacaatga gcttcgagta  361 gcaccagaag agcaccccac cctgctcaca gaggcccccc taaaccccaa agcaaacaga  421 gagaagatga cccagatcat gttcgaaacc ttcaatgtcc ctgccatgta tgttgctatt  481 caggctgtgc tctcactcta tgcatccggc cgtaccacag gcatcgttct ggattcgggg  541 gatggcgtca cccacaatgt ccccatctat gagggctatg cactgcccca tgccatcatg  601 cgtcttgacc tggctggacg ggatctcaca gactacctca tgaagattct cacagaaaga  661 ggctattcct ttgtgaccac agctgagaga gaaattgtac gagacatcaa ggagaagctg  721 tgctatgtag ccctggattt cgagaatgag atggccacag cagcttcatc ttcttccctg  781 gagaaaagct acgagttgcc tgatgggcag gtcatcacta ttggcaacga gcgcttccgc  841 tgcccggaga ccctcttcca gccttccttc attggcatgg agtcagctgg aattcatgaa  901 acaacataca attccatcat gaagtgtgac attgacatcc gcaaagattt gtatgctaac  961 aatgtcctct ctgggggcac taccatgtac cctggcattg ctgacaggat gcagaaggaa 1021 atcacagcct tggctcccag caccatgaag atcaagatta tcgctcctcc tgagcggaag 1081 tactcagtct ggattggcgg ctccatcctg gcctctctct ccaccttcca gcaaatgtgg 1141 atcagcaagc cagagtatga tgaggcaggg ccctccattg tccacaggaa atgcttc taa 1201 agtcagaggg ccttctctgg ggatccccac aagactgctg tcaccagcca cagatcatta 1261 aaaccttcaa gccgaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 

The amino acid sequence of PAI-1 is provided by GenBank Accession No. P05121.1 and is provided below.

(SEQ ID NO: 21)   1 mqmspaltcl vlglalvfge gsavhhppsy vahlasdfgv rvfqqvaqas kdrnvvfspy  61 gvasvlamlq lttggetqqq iqaamgfkid dkgmapalrh lykelmgpwn kdeisttdai 121 fvqrdlklvg gfmphffrlf rstvkqvdfs everarfiin dwvkthtkgm isnllgkgav 181 dqltrlvlvn alyfngqwkt pfpdssthrr lfhksdgstv svpmmaqtnk fnytefttpd 241 ghyydilelp yhgdtlsmfi aapyekevpl saltnilsaq lishwkgnmt rlprllvlpk 301 fsletevdlr kplenlgmtd mfrqfqadft slsdgeplhv aqalqkvkie vnesgtvass 361 stavivsarm apeeiimdrp flfvvrhnpt gtvlfmgqvm ep 

The mRNA sequence of PAI-1 is provided by GenBank Accession No. M16006.1 and is provided below. The start and stop codons are in bold and underlined.

(SEQ ID NO: 22)    1 gaattcctgc agctcagcag ccgccgccag agcaggacga accgccaatc gcaaggcacc   61 tctgagaact tcagg atg ca gatgtctcca gccctcacct gcctagtcct gggcctggcc  121 cttgtctttg gtgaagggtc tgctgtgcac catcccccat cctacgtggc ccacctggcc  181 tcagacttcg gggtgagggt gtttcagcag gtggcgcagg cctccaagga ccgcaacgtg  241 gttttctcac cctatggggt ggcctcggtg ttggccatgc tccagctgac aacaggagga  301 gaaacccagc agcagattca agcagctatg ggattcaaga ttgatgacaa gggcatggcc  361 cccgccctcc ggcatctgta caaggagctc atggggccat ggaacaagga tgagatcagc  421 accacagacg cgatcttcgt ccagcgggat ctgaagctgg tccagggctt catgccccac  481 ttcttcaggc tgttccggag cacggtcaag caagtggact tttcagaggt ggagagagcc  541 agattcatca tcaatgactg ggtgaagaca cacacaaaag gtatgatcag caacttgctt  601 gggaaaggag ccgtggacca gctgacacgg ctggtgctgg tgaatgccct ctacttcaac  661 ggccagtgga agactccctt ccccgactcc agcacccacc gccgcctctt ccacaaatca  721 gacggcagca ctgtctctgt gcccatgatg gctcagacca acaagttcaa ctatactgag  781 ttcaccacgc ccgatggcca ttactacgac atcctggaac tgccctacca cggggacacc  841 ctcagcatgt tcattgctgc cccttatgaa aaagaggtgc ctctctctgc cctcaccaac  901 attctgagtg cccagctcat cagccactgg aaaggcaaca tgaccaggct gccccgcctc  961 ctggttctgc ccaagttctc cctggagact gaagtcgacc tcaggaagcc cctagagaac 1021 ctgggaatga ccgacatgtt cagacagttt caggctgact tcacgagtct ttcagaccaa 1081 gagcctctcc acgtcgcgca ggcgctgcag aaagtgaaga tcgaggtgaa cgagagtggc 1141 acggtggcct cctcatccac agctgtcata gtctcagccc gcatggcccc cgaggagatc 1201 atcatggaca gacccttcct ctttgtggtc cggcacaacc ccacaggaac agtccttttc 1261 atgggccaag tgatggaacc c tga ccctgg ggaaagacgc cttcatctgg gacaaaactg 1321 gagatgcatc gggaaagaag aaactccgaa gaaaagaatt ttagtgttaa tgactctttc 1381 tgaaggaaga gaagacattt gccttttgtt aaaagatggt aaaccagatc tgtctccaag 1441 accttggcct ctccttggag gacctttagg tcaaactccc tagtctccac ctgagaccct 1501 gggagagaag tttgaagcac aactccctta aggtctccaa accagacggt gacgcctgcg 1561 ggaccatctg gggcacctgc ttccacccgt ctctctgccc actcgggtct gcagacctgg 1621 ttcccactga ggccctttgc aggatggaac tacggggctt acaggagctt ttgtgtgcct 1681 ggtagaaact atttctgttc cagtcacatt gccatcactc ttgtactgcc tgccaccgcg 1741 gaggaggctg gtgacaggcc aaaggccagt ggaagaaaca ccctttcatc tcagagtcca 1801 ctgtggcact ggccacccct ccccagtaca ggggtgctgc aggtggcaga gtgaatgtcc 1861 cccatcatgt ggcccaactc tcctggcctg gccatctccc tccccagaaa cagtgtgcat 1921 gggttatttt ggagtgtagg tgacttgttt actcattgaa gcagatttct gcttcctttt 1981 atttttatag gaatagagga agaaatgtca gatgcgtgcc cagctcttca ccccccaatc 2041 tcttggtggg gaggggtgta cctaaatatt tatcatatcc ttgcccttga gtgcttgtta 2101 gagagaaaga gaactactaa ggaaaataat attatttaaa ctcgctccta gtgtttcttt 2161 gtggtctgtg tcaccgtatc tcaggaagtc cagccacttg actggcacac acccctccgg 2221 acatccagcg tgacggagcc cacactgcca ccttgtggcc gcctgagacc ctcgcgcccc 2281 ccgcgccccc cgcgcccctc tttttcccct tgatggaaat tgaccataca atttcatcct 2341 ccttcagggg atcaaaagga cggagtgggg ggacagagac tcagatgagg acagagtggt 2401 ttccaatgtg ttcaatagat ttaggagcag aaatgcaagg ggctgcatga cctaccagga 2461 cagaactttc cccaattaca gggtgactca cagccgcatt ggtgactcac ttcaatgtgt 2521 catttccggc tgctgtgtgt gagcagtgga cacgtgaggg gggggtgggt gagagagaca 2581 ggcagctcgg attcaactac cttagataat atttctgaaa acctaccagc cagagggtag 2641 ggcacaaaga tggatgtaat gcactttggg aggccaaggc gggaggattg cttgagccca 2701 ggagttcaag accagcctgg gcaacatacc aagacccccg tctctttaaa aatatatata 2761 ttttaaatat acttaaatat atatttctaa tatctttaaa tatatatata tattttaaag 2821 accaatttat gggagaattg cacacagatg tgaaatgaat gtaatctaat agaagc 

The protein sequence encoding human TGFβ1 is provided by GenBank Accession No:

P01137.2 (incorporated herein by reference), and the sequence is shown below.

(SEQ ID NO: 23)   1 mppsglrlll lllpllwllv ltpgrpaagl stcktidmel vkrkrieair gqilsklrla  61 sppsqgevpp gplpeavlal ynstrdrvag esaepepepe adyyakevtr vlmvethnei 121 ydkfkqsths iymffntsel reavpepvll sraelrllrl klkveqhvel yqkysnnswr 181 ylsnrllaps dspewlsfdv tgvvrqwlsr ggeiegfrls ahcscdsrdn tlqvdingft 241 tgrrgdlati hgmnrpf111 matpleraqh lqssrhrral dtnycfsste kncovrqlyi 301 dfrkdlgwkw ihepkgyhan fclgpcpyiw sldtgyskvl alynqhnpga saapccvpqa 361 leplpivyyv grkpkveqls nmivrsckcs

The mRNA sequence encoding human TGFβ1 is provided by GenBank Accession No:

NM_000660.5 (incorporated herein by reference), and the sequence is shown below.

(SEQ ID NO: 24)    1 agccggtccc cgccgccgcc gcccttcgcg ccctgggcca tctccctccc acctccctcc   61 gcggagcagc cagacagcga gggccccggc cgggggcagg ggggacgccc cgtccggggc  121 acccccccgg ctctgagccg cccgcggggc cggcctcggc ccggagcgga ggaaggagtc  181 gccgaggagc agcctgaggc cccagagtct gagacgagcc gccgccgccc ccgccactgc  241 ggggaggagg gggaggagga gcgggaggag ggacgagctg gtcgggagaa gaggaaaaaa  301 acttttgaga cttttccgtt gccgctggga gccggaggcg cggggacctc ttggcgcgac  361 gctgccccgc gaggaggcag gacttgggga ccccagaccg cctccctttg ccgccgggga  421 cgcttgctcc ctccctgccc cctacacggc gtccctcagg cgcccccatt ccggaccagc  481 cctcgggagt cgccgacccg gcctcccgca aagacttttc cccagacctc gggcgcaccc  541 cctgcacgcc gccttcatcc ccggcctgtc tcctgagccc ccgcgcatcc tagacccttt  601 ctcctccagg agacggatct ctctccgacc tgccacagat cccctattca agaccaccca  661 ccttctggta ccagatcgcg cccatctagg ttatttccgt gggatactga gacacccccg  721 gtccaagcct cccctccacc actgcgccct tctccctgag gacctcagct ttccctcgag  781 gccctcctac cttttgccgg gagaccccca gcccctgcag gggcggggcc tccccaccac  841 accagccctg ttcgcgctct cggcagtgcc ggggggcgcc gcctccccca tgccgccctc  901 cgggctgcgg ctgctgccgc tgctgctacc gctgctgtgg ctactggtgc tgacgcctgg  961 ccggccggcc gcgggactat ccacctgcaa gactatcgac atggagctgg tgaagcggaa 1021 gcgcatcgag gccatccgcg gccagatcct gtccaagctg cggctcgcca gccccccgag 1081 ccagggggag gtgccgcccg gcccgctgcc cgaggccgtg ctcgccctgt acaacagcac 1141 ccgcgaccgg gtggccgggg agagtgcaga accggagccc gagcctgagg ccgactacta 1201 cgccaaggag gtcacccgcg tgctaatggt ggaaacccac aacgaaatct atgacaagtt 1261 caagcagagt acacacagca tatatatgtt cttcaacaca tcagagctcc gagaagcggt 1321 acctgaaccc gtgttgctct cccgggcaga gctgcgtctg ctgaggctca agttaaaagt 1381 ggagcagcac gtggagctgt accagaaata cagcaacaat tcctggcgat acctcagcaa 1441 ccggctgctg gcacccagcg actcgccaga gtggttatct tttgatgtca ccggagttgt 1501 gcggcagtgg ttgagccgtg gaggggaaat tgagggcttt cgccttagcg cccactgctc 1561 ctgtgacagc agggataaca cactgcaagt ggacatcaac gggttcacta ccggccgccg 1621 aggtgacctg gccaccattc atggcatgaa ccggcctttc ctgcttctca tggccacccc 1681 gctggagagg gcccagcatc tgcaaagctc ccggcaccgc cgagccctgg acaccaacta 1741 ttgcttcagc tccacggaga agaactgctg cgtgcggcag ctgtacattg acttccgcaa 1801 ggacctcggc tggaagtgga tccacgagcc caagggctac catgccaact tctgcctcgg 1861 gccctgcccc tacatttgga gcctggacac gcagtacagc aaggtcctgg ccctgtacaa 1921 ccagcataac ccgggcgcct cggcggcgcc gtgctgcgtg ccgcaggcgc tggagccgct 1981 gcccatcgtg tactacgtgg gccgcaagcc caaggtggag cagctgtcca acatgatcgt 2041 gcgctcctgc aagtgcagct gaggtcccgc cccgccccgc cccgccccgg caggcccggc 2101 cccaccccgc cccgcccccg ctgccttgcc catgggggct gtatttaagg acacccgtgc 2161 cccaagccca cctggggccc cattaaagat ggagagagga ctgcggatct ctgtgtcatt 2221 gggcgcctgc ctggggtctc catccctgac gttcccccac tcccactccc tctctctccc 2281 tctctgcctc ctcctgcctg tctgcactat tcctttgccc ggcatcaagg cacaggggac 2341 cagtggggaa cactactgta gttagatcta tttattgagc accttgggca ctgttgaagt 2401 gccttacatt aatgaactca ttcagtcacc atagcaacac tctgagatgc agggactctg 2461 ataacaccca ttttaaaggt gaggaaacaa gcccagagag gttaagggag gagttcctgc 2521 ccaccaggaa cctgctttag tgggggatag tgaagaagac aataaaagat agtagttcag 2581 gcc

In other embodiments, the population of fibroblasts comprises a genetically modified fibroblast.

As described above, the composition optionally comprises a bioactive composition. The bioactive composition decreases inflammation, increases vascular regeneration, increases muscular regeneration, and/or promote skin regeneration. Exemplary bioactive compositions are described above.

The number of cells in a composition and the mode of administration may vary depending on the site and condition being treated (e.g., location of a wound or size of a wound). As non-limiting examples, in accordance with the present invention, a device seeded with about 10-500×10⁶ fibroblasts is administered to a subject (e.g., diabetic subject) to effect wound healing. A skilled practitioner can modulate the amounts and methods of fibroblast-based treatments according to requirements, limitations, and/or optimizations determined for each case.

In some embodiments, the scaffold composition comprises between about 10×10⁶ and 600×10⁶ viable cells (e.g., fibroblasts). In some cases, the cells (e.g., fibroblasts) are seeded at a concentration of about 1×10³ to 1×10⁸ cells/ml (e.g., about 5×10³ to 5×10⁷ cells/ml, or about 1×10⁴ to 1×10⁷ cells/ml) into the device. For example, the cells are seeded in a device having a volume of 1-500 uL (e.g., 10-250 uL, 20-100 uL, or 40-60 uL, or about 50 uL). The dose of the device to be delivered to the subject depends on the magnitude of the injury or diseased area, e.g., one milliliter of gel for a relatively small wound and up to 50 mls of gel for a large wound. In some examples, the device has a volume of 1-500 uL (e.g., 10-250 uL, 20-100 uL, or 40-60 uL, or about 50 uL).

The composition preferably has less than 0.5 EU/ml of endotoxin and no bacterial or fungal growth.

The present invention also features a method of treating a wound in a patient in need thereof comprising administering a composition described herein. In some cases, the patient suffers from diabetes and/or a wound (e.g., located in an extremity of the patient). Exemplary extremities include arms, legs, feet, hands, fingers, and toes. For example, the patient suffers from an ulcer, e.g., a foot ulcer. Exemplary ulcers are at least about 25 mm, 50 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, or greater in diameter.

The method provides compositions in which the population of fibroblasts includes an autologous, allogeneic, or xenogeneic fibroblast. For example, the population of fibroblasts comprises at least 10% autologous fibroblasts (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more); at least 10% allogeneic fibroblasts (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more); or at least 10% xenogeneic fibroblasts (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more).

The described the devices are administered to the tissues of the recipient organism of interest, including humans and non-human animals.

Devices of the present invention are administered or implanted orally, systemically, sub-or trans-cutaneously, as an arterial stent, surgically, or via injection. In some examples, the devices described herein are administered by routes such as injection (e.g., subcutaneous, intravenous, intracutaneous, percutaneous, or intramuscular) or implantation.

In one embodiment, administration of the device is mediated by injection or implantation into a wound or a site adjacent to the wound. For example, the wound is external or internal.

The invention will be further illustrated in the following non-limiting examples.

EXAMPLES Example 1 Effect of Fibroblasts Derived from a Site Adjacent to a Diabetic Foot Ulcer on Wound Healing

A mouse cutaneous wound-healing model was used to characterize the in vivo wound-healing potential of isolated fibroblasts. Specifically, mouse models were used to determine the effect of various populations of fibroblasts on wound healing in non-diabetic and diabetic mice.

Alginate hydrogels were used as exemplary material systems and delivery devices, as the physical, chemical, and biological properties of alginate gels can be readily manipulated to develop gels with a range of properties. For example, the gel, e.g., injectable gel, can be introduced directly into the tissue at or surrounding the wound, via needle or syringe, where the transplated cells aid in upregulating a host healing response.

High G-block-containing alginate, MVG (M:G=40:60; MW ˜250 kDa; ProNova Biomedical, Oslo, Norway) was used to also obtain low-molecular alginate (MW ˜50 kDa) by gamma irradiation (3 MRad) (EH&S Lab at MIT, Cambridge, Mass.). To facilitate degradation and cell release, 1% of the sugar residues in the alginate chains were oxidized using sodium periodate (Sigma-Aldrich) and the alginate was dialyzed and lyophilized. Alginate chains were further modified to contain RGD-binding domains (GGGGRGDSH (SEQ ID NO: 29), Peptides 2.0, Virginia; two RGD per HMW chain or per five LMW chain) using carbodiimide chemistry, dialyzed, and lyophilized. The final concentration of alginate gels was 2% w/v (HMW:LMW=25:75) in serum-free DMEM. Gels contained le⁶ cells per 60 mL and were ionically cross-linked with 4% v/v 1.22M calcium sulfate solution. See, Kong et al., 2004 Biomacromolecules 5, 1720, incorporated herein by reference.

Sixteen-week-old C57BL6 male mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized, and two 6-mm full-thickness skin punch biopsies were obtained from the shaved dorsum of the animals. Fibroblasts were combined with an alginate-based hydrogel formulation described above. A total of 60 mL of blank hydrogel or cell-loaded hydrogel (1×10⁶ cells/wound) was injected intradermally into three sites along the wound margins immediately after wound creation (day 0).

The study groups were as follows. Both groups of mice (i.e., diabetic and non-diabetic) were treated with the following four treatments: a) alginate hydrogel-containing device alone (Hydrogel); b) alginate hydrogel-containing device that contains fibroblasts originating from the skin area adjacent to an existing chronic diabetic foot ulcer (F-DFU); c) alginate hydrogel-containing device that contains fibroblasts originating from the skin of the dorsum of the foot of a diabetic foot ulcer (F-non-DFU); d) alginate hydrogel-containing device that contains fibroblasts originating from the skin of the dorsum of the foot of a non-diabetic subject with no serious health issues (F-control). Healing was monitored over 10 days by daily wound tracing and is expressed as mean percentage of original wound size (day 0). Mice were euthanized at 10 days postwounding in order to collect wound tissue for histological analysis.

In non-diabetic mice, the best wound healing (i.e., the fastest reduction in wound size) was achieved by the alginate hydrogel that contained fibroblasts from the non-diabetic subject (FIG. 1). See, Maione et al., 2015 Tissue Engineering, 21(5): 499-508, incorporated herein by reference. In contrast, in diabetic mice, the best wound healing (i.e., the fastest reduction in wound size) was achieved by the hydrogel that contained fibroblasts taken from the skin area in or adjacent to a diabetic foot ulcer (FIG. 2). Thus, these results demonstrate that hydrogels comprising fibroblasts derived from diabetic foot ulcers lead to significantly improved diabetic wound, i.e., foot ulcer, healing compared to nondiabetic, nonulcerated foot-derived fibroblasts. As such, these results demonstrate that these diabetic foot ulcer derived fibroblasts that were previously incapable of healing the diabetic ulcer were “reprogrammed” in the presence of the hydrogel delivery vehicle to effectively heal the diabetic ulcer.

Example 2 Microarray Analysis: Diabetic Foot Ulcer-Derived Fibroblasts (DFUF) and Diabetic Non-Ulcerated Foot-Derived Fibroblasts (DFF) Exhibited Differential Gene Expression Profiles Compared to Non-Ulcerated Foot-Derived Fibroblasts (NFF)

An in vivo-like experimental model that more closely mimics the diabetic microenvironment was developed to understand how altered extracellular matrix (ECM) contributes to impaired diabetic foot ulcer (DFU). Specifically, three-dimensional (3D), skin-like tissues were developed to study the production and assembly of ECM by fibroblasts isolated from DFU (DFUFs), site-matched, non-ulcerated, diabetic skin (DFFs) and site-matched, non-ulcerated, non-diabetic skin (NFFs). This self-assembled ECM mimics the composition of early granulation tissue and is useful in studying fibroblast function during normal and abnormal healing (Maione et al., 2015 Tissue Engineering, 21(5): 499-508, incorporated herein by reference).

As described herein, this model was used to identify that phenotypic differences in ECM organization in DFU vs. DFF and DFU vs. NFF controls were related to variable responses to TGFB and in the production of fibronectin. As described below, fibronectin is an important marker expressed by DFU-derived fibroblasts and is linked to the altered wound healing manifested by these cells in vivo. These responses are also linked to the metabolic memory that cells exposed to a hyperglycemic environment manifest through changes in their ECM production and response to growth factors. The following markers are of relevance to the DFU phenotype in 2D culture and in 3D self-assembled tissues.

Fibronectin is elevated in some tissues and organs in diabetes (e.g., kidney), but prior to the invention described herein, an elevation of fibronectin has not been identified in DFU. Additionally, prior to the invention described herein, fibronectin was not associated as a marker of metabolic memory of a diabetic wound physiological microenvironment. The results described herein show that elevated fibronectin is a marker indicating that DFU-derived fibroblasts have an altered ECM phenotype which affects their ability to support proper wound healing. While there was little or no difference in fibronectin expression in a 2D system, DFUFs produced and assembled ECM in 3D tissues and were significantly enriched in fibronectin as compared to NFFs as seen by Western blot. The 3D model described herein is a complex tissue system that provides more physiologically relevant information by mimicking the in vivo environment as compared to a 2D system. These results demonstrate that the elevated production of fibronectin in DFU-derived fibroblasts (DFUF) has important functional consequences in alterations seen in chronic wound repair. These results also demonstrate that fibronectin is an important marker of DFU and is relevant to metabolic memory.

For example, the population of fibroblasts derived from diabetic foot ulcers expresses fibronectin at a level at least 1.1 fold more than nondiabetic, nonulcerated foot-derived fibroblasts, e.g., at least 1.2 fold more, at least 1.3 fold more, at least 1.4 fold more, at least 1.5 fold more, at least 1.6 fold more, at least 1.7 fold more, at least 1.8 fold more, at least 1.9 fold more, at least 2 fold more, at least 3 fold more, at least 4 fold more, at least 5 fold more, at least 6 fold more, at least 7 fold more, at least 8 fold more, at least 9 fold more, at least 10 fold more, at least 11 fold more, at least 12 fold more, at least 13 fold more, at least 14 fold more, or at least 15 fold more.

Fibronectin needs to be cleared/decreased before normal repair can proceed. The sustained expression and deposition of fibronectin prevents the progression of normal wound healing, but prior to the invention described herein, this was not studied specifically in DFU.

Smooth muscle actin is elevated in DFUFs treated with transforming growth factor beta (TGF-β). As described herein, DFUFs responded to TGF-I3 stimulation in 3D self-assembled tissues to activate myofibroblast phenotype through increased production of smooth muscle actin (SMA).

While DFFs and NFFs increased their fibronectin and ED-A fibronectin production in response to TGF-β, DFUFs down regulated the production of these ECM proteins. This decrease in fibronectin is a positive step towards wound healing as it needs to be cleared/decreased before normal repair can proceed. This suggests that TGF-I3 signaling in the DFU environment may be abnormal and may not suppress fibronectin, which could be a function of metabolic memory that is maintaining fibronectin in an elevated state.

As described herein, the results identified a trend of induction of miR-21-5p and miR-143-3p in DFUFs as compared to NFFs, whereas a trend of suppression of miR-29c-3p and miR-155-5p were shown in both DFUFs, and DFFs when compared to NFFs. This suggests these miRs are markers by which ECM and TGFβ signaling can be differentially regulated between DFUFs, DFFs and NFFs.

The mRNA sequence encoding miR-21-5p is provided by GenBank Accession No: MIMAT0000076 (incorporated herein by reference; UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO: 25)). The mRNA sequence encoding miR-143-3p is provided by GenBank Accession No: MIMAT0000435 (incorporated herein by reference; UGAGAUGAAGCACUGUAGCUC (SEQ ID NO: 26)). The mRNA sequence encoding miR-29c-3p is provided by GenBank Accession No: MIMAT0000681 (incorporated herein by reference; UAGCACCAUUUGAAAUCGGUUA (SEQ ID NO: 27)). The mRNA sequence encoding miR-155-5p is provided by GenBank Accession No: MIMAT0000646 (incorporated herein by reference; UUAAUGCUAAUCGUGAUAGGGGU (SEQ ID NO: 28)).

To examine gene expression differences between diabetic foot ulcer-derived fibroblasts (DFUF), diabetic non-ulcerated foot-derived fibroblasts (DFF), and nonulcerated foot-derived fibroblasts (NFF), microarray analysis was conducted on twelve cell lines using Illumina's human BeadChip® array profiling over 47,000 transcripts (HumanHT-12 v4 Expression BeadChip Kit; Illumina, Inc; San Diego, Calif.). Unsupervised hierarchal clustering using Euclidian distance and Ward linkage was conducted to determine global differences in mRNA expression. This analysis identified 170 differentially expressed genes between DFFs and NFFs, 115 differentially expressed genes between DFUF and NFF and 58 differentially expressed genes between DFUF and DFF. Examination of differentially expressed genes revealed enrichment, i.e., increased expression, in ECM-related gene terms using gene set enrichment analysis. Specifically, Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GORILLA) was used for the analysis of gene set enrichment (cbl-gorilla.cs.technion.ac.il/), while “Microarray R Us” and “R” was used to analyze the microarray data (norris.usc.libguides.com/MicroarrayRUS).

For example, the genes in the table below or the miRs described above are enriched (i.e., upregulated) in fibroblasts derived from diabetic foot ulcers at a level at least 1.1 fold more than nondiabetic, nonulcerated foot-derived fibroblasts, e.g., at least 1.2 fold more, at least 1.3 fold more, at least 1.4 fold more, at least 1.5 fold more, at least 1.6 fold more, at least 1.7 fold more, at least 1.8 fold more, at least 1.9 fold more, at least 2 fold more, at least 3 fold more, at least 4 fold more, at least 5 fold more, at least 6 fold more, at least 7 fold more, at least 8 fold more, at least 9 fold more, at least 10 fold more, at least 11 fold more, at least 12 fold more, at least 13 fold more, at least 14 fold more, at least 15 fold more, at least 20 fold more, at least 30 fold more, at least 40 fold more, at least 50 fold more, at least 60 fold more, at least 70 fold more, at least 80 fold more, at least 90 fold more, or at least 100 fold more. Alternatively, the genes in the table below or the miRs described above are downregulated in fibroblasts derived from diabetic foot ulcers at a level at least 1.1 fold less than nondiabetic, nonulcerated foot-derived fibroblasts, e.g., at least 1.2 fold less, at least 1.3 fold less, at least 1.4 fold less, at least 1.5 fold less, at least 1.6 fold less, at least 1.7 fold less, at least 1.8 fold less, at least 1.9 fold less, at least 2 fold less, at least 3 fold less, at least 4 fold less, at least 5 fold less, at least 6 fold less, at least 7 fold less, at least 8 fold less, at least 9 fold less, at least 10 fold less, at least 11 fold less, at least 12 fold less, at least 13 fold less, at least 14 fold less, at least 15 fold less, at least 20 fold less, at least 30 fold less, at least 40 fold less, at least 50 fold less, at least 60 fold less, at least 70 fold less, at least 80 fold less, at least 90 fold less, or at least 100 fold less.

This includes enrichment in gene ontology terms related to glucose metabolism supporting differences between the fibroblast groups based on diabetes status. Additionally, other processes related to wound healing, such as leukocyte chemotaxis, cell migration, cytokine production and angiogenesis, were also enriched. Several gene terms pertaining to ECM production and organization were significantly enriched in each of the three categories; biological processes, molecular functions and cellular components. The results are shown in the tables below.

Bio Process GO Term Description P-value FDR q-value Enrichment Genes GO:0019682 glyceraldehyde- 3.70E−04 3.23E−02 59.62 [TPI1 - triosephosphate isomerase 1, TKT - 3-phosphate transketolase] metabolic process GO:2000353 positive 2.13E−04 2.30E−02 24.39 [AKR1C3 - aldo-keto reductase family 1, regulation of member c3, RGCC - regulator of cell endothelial cycle, COL18A1 - collagen, type xviii, cell apoptotic alpha 1] process GO:0002686 negative 5.15E−05 9.66E−03 18.83 [GREM1 - gremlin 1, dan family bmp regulation antagonist, APOD - apolipoprotein d, of leukocyte HMOX1 - heme oxygenase (decycling) 1, migration CCL2 - chemokine (c-c motif) ligand 2] GO:0048247 lymphocyte 6.92E−04 4.99E−02 16.77 [CXCL16 - chemokine (c-x-c motif) chemotaxis ligand 16, GAS6 - growth arrest-specific 6, CCL2 - chemokine (c-c motif) ligand 2] GO:2000404 regulation of 1.14E−04 1.61E−02 15.55 [APOD - apolipoprotein d, TNFRSF14 - T cell tumor necrosis factor receptor superfamily, migration member 14, PYCARD - pyd and card domain containing, RIPK3 - receptor- interacting serine-threonine kinase 3] GO:0006096 glycolytic 4.50E−07 4.41E−04 14.56 [PFKFB4 - 6-phosphofructo-2- process kinase/fructose-2,6-biphosphatase 4, PGK1 - phosphoglycerate kinase 1, TPI1 - triosephosphate isomerase 1, PFKP - phosphofructokinase, platelet, PGM1 - phosphoglucomutase 1, PGAM1 - phosphoglycerate mutase 1 (brain), ENO2 - enolase 2 (gamma, neuronal)] GO:2000401 regulation of 2.62E−05 5.85E−03 13.97 [APOD - apolipoprotein d, TNFRSF14 - lymphocyte tumor necrosis factor receptor superfamily, migration member 14, CCL2 - chemokine (c-c motif) ligand 2, PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3] GO:1901655 cellular 2.89E−04 2.84E−02 12.34 [TNFSF4 - tumor necrosis factor (ligand) response superfamily, member 4, GAS6 - growth to ketone arrest-specific 6, ASS1 - argininosuccinate synthase 1, AQP1 - aquaporin 1 (colton blood group)] GO:0050710 negative 3.31E−04 3.08E−02 11.92 [RGCC - regulator of cell cycle, TNFSF4 - regulation of tumor necrosis factor (ligand) cytokine superfamily, member 4, GAS6 - growth secretion arrest-specific 6, SRGN - serglycin] GO:2000351 regulation of 3.77E−04 3.24E−02 11.54 [RGCC - regulator of cell cycle, AKR1C3 - endothelial aldo-keto reductase family 1, member c3, cell apoptotic COL18A1 - collagen, type xviii, alpha 1, process GAS6 - growth arrest-specific 6] GO:0030199 collagen fibril 7.95E−05 1.30E−02 11.18 [GREM1 - gremlin 1, dan family bmp organization antagonist, FOXC1 - forkhead box c1, SFRP2 - secreted frizzled-related protein 2, LUM - lumican, COL11A1 - collagen, type xi, alpha 1] GO:0006081 cellular 1.41E−04 1.80E−02 9.94 [AKR1C3 - aldo-keto reductase family 1, aldehyde member c3, ALDH3A1 - aldehyde metabolic dehydrogenase 3 family, member a1, process ALDH3A2 - aldehyde dehydrogenase 3 family, member a2, TPI1 - triosephosphate isomerase 1, TKT - transketolase] GO:0006094 gluconeogenesis 1.41E−04 1.82E−02 9.94 [PGK1 - phosphoglycerate kinase 1, TPI1 - triosephosphate isomerase 1, PGM1 - phosphoglucomutase 1, PGAM1 - phosphoglycerate mutase 1 (brain), ENO2 - enolase 2 (gamma, neuronal)] GO:0071347 cellular 1.41E−04 1.84E−02 9.94 [PTGIS - prostaglandin i2 (prostacyclin) response to synthase, IL1R1 - interleukin 1 receptor, interleukin-1 type i, CCL2 - chemokine (c-c motif) ligand 2, KLF2 - kruppel-like factor 2 (lung), PYCARD - pyd and card domain containing] GO:0006090 pyruvate 1.30E−05 3.68E−03 8.94 [PFKFB4 - 6-phosphofructo-2- metabolic kinase/fructose-2,6-biphosphatase 4, PGK1 - process phosphoglycerate kinase 1, TPI1 - triosephosphate isomerase 1, PFKP - phosphofructokinase, platelet, PGM1 - phosphoglucomutase 1, PGAM1 - phosphoglycerate mutase 1 (brain), ENO2 - enolase 2 (gamma, neuronal)] GO:0030574 collagen 1.88E−05 4.69E−03 8.46 [CTSK - cathepsin k, COL8A2 - catabolic collagen, type viii, alpha 2, COL23A1 - process collagen, type xxiii, alpha 1, COL4A1 - collagen, type iv, alpha 1, COL18A1 - collagen, type xviii, alpha 1, COL11A1 - collagen, type xi, alpha 1, COL15A1 - collagen, type xv, alpha 1] GO:0032963 collagen 3.98E−05 7.92E−03 7.54 [CTSK - cathepsin k, COL23A1 - metabolic collagen, type xxiii, alpha 1, COL8A2 - process collagen, type viii, alpha 2, COL4A1 - collagen, type iv, alpha 1, COL18A1 - collagen, type xviii, alpha 1, COL11A1 - collagen, type xi, alpha 1, COL15A1 - collagen, type xv, alpha 1] GO:0002685 regulation of 3.17E−06 1.39E−03 7.52 [GREM1 - gremlin 1, dan family bmp leukocyte antagonist, THBS4 - thrombospondin 4, migration APOD - apolipoprotein d, TNFRSF14 - tumor necrosis factor receptor superfamily, member 14, HMOX1 - heme oxygenase (decycling) 1, GAS6 - growth arrest- specific 6, CCL2 - chemokine (c-c motif) ligand 2, PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3] GO:1901654 response to 2.03E−05 4.98E−03 6.95 [TNFSF4 - tumor necrosis factor (ligand) ketone superfamily, member 4, DUSP1 - dual specificity phosphatase 1, SLIT3 - slit homolog 3 (drosophila), GAS6 - growth arrest-specific 6, CA9 - carbonic anhydrase ix, ASS1 - argininosuccinate synthase 1, CCL2 - chemokine (c-c motif) ligand 2, AQP1 - aquaporin 1 (colton blood group)] GO:0032675 regulation of 2.74E−04 2.77E−02 6.71 [TNFSF4 - tumor necrosis factor (ligand) interleukin-6 superfamily, member 4, ADORA2B - production adenosine a2b receptor, GAS6 - growth arrest-specific 6, KLF2 - kruppel-like factor 2 (lung), PYCARD - pyd and card domain containing, CARD9 - caspase recruitment domain family, member 9] GO:0071456 cellular 1.08E−04 1.58E−02 6.45 [CITED2 - cbp/p300-interacting response to transactivator, with glu/asp-rich carboxy- hypoxia terminal domain, 2, RGCC - regulator of cell cycle, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, PTGIS - prostaglandin i2 (prostacyclin) synthase, HMOX1 - heme oxygenase (decycling) 1, CA9 - carbonic anhydrase ix, AQP1 - aquaporin 1 (colton blood group)] GO:0036294 cellular 1.23E−04 1.68E−02 6.32 [CITED2 - cbp/p300-interacting response to transactivator, with glu/asp-rich carboxy- decreased terminal domain, 2, RGCC - regulator of oxygen levels cell cycle, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, PTGIS - prostaglandin i2 (prostacyclin) synthase, HMOX1 - heme oxygenase (decycling) 1, CA9 - carbonic anhydrase ix, AQP1 - aquaporin 1 (colton blood group)] GO:0016052 carbohydrate 4.80E−05 9.27E−03 6.17 [PFKFB4 - 6-phosphofructo-2- catabolic kinase/fructose-2,6-biphosphatase 4, PGK1 - process phosphoglycerate kinase 1, TPI1 - triosephosphate isomerase 1, PFKP - phosphofructokinase, platelet, PGM1 - phosphoglucomutase 1, PGAM1 - phosphoglycerate mutase 1 (brain), TKT - transketolase, ENO2 - enolase 2 (gamma, neuronal)] GO:0043627 response to 1.88E−06 9.98E−04 6.15 [CITED2 - cbp/p300-interacting estrogen transactivator, with glu/asp-rich carboxy- terminal domain, 2, CTGF - connective tissue growth factor, WFDC1 - wap four- disulfide core domain 1, GSTM3 - glutathione s-transferase mu 3 (brain), DUSP1 - dual specificity phosphatase 1, RCAN1 - regulator of calcineurin 1, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, ASS1 - argininosuccinate synthase 1, AQP1 - aquaporin 1 (colton blood group), TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b] GO:0051224 negative 5.76E−05 1.05E−02 6.01 [RGCC - regulator of cell cycle, TNFSF4 - regulation of tumor necrosis factor (ligand) protein superfamily, member 4, APOD - transport apolipoprotein d, NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, SOX4 - sry (sex determining region y)-box 4, GAS6 - growth arrest-specific 6, SRGN - serglycin, AXIN2 - axin 2] GO:0090090 negative 5.49E−04 4.21E−02 5.9 [PRICKLE1 - prickle homolog 1 regulation of (drosophila), GREM1 - gremlin 1, dan canonical Wnt family bmp antagonist, SFRP2 - secreted signaling frizzled-related protein 2, DKK3 - pathway dickkopf wnt signaling pathway inhibitor 3, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), AXIN2 - axin 2] GO:0071453 cellular 1.99E−04 2.21E−02 5.85 [CITED2 - cbp/p300-interacting response to transactivator, with glu/asp-rich carboxy- oxygen levels terminal domain, 2, RGCC - regulator of cell cycle, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, PTGIS - prostaglandin i2 (prostacyclin) synthase, HMOX1 - heme oxygenase (decycling) 1, CA9 - carbonic anhydrase ix, AQP1 - aquaporin 1 (colton blood group)] GO:0032387 negative 6.16E−04 4.59E−02 5.77 [APOD - apolipoprotein d, HMOX1 - regulation of heme oxygenase (decycling) 1, NFKBIA - intracellular nuclear factor of kappa light polypeptide transport gene enhancer in b-cells inhibitor, alpha, SOX4 - sry (sex determining region y)-box 4, GAS6 - growth arrest-specific 6, AXIN2 - axin2] GO:0030336 negative 1.37E−05 3.70E−03 5.56 [CITED2 - cbp/p300-interacting regulation of transactivator, with glu/asp-rich carboxy- cell migration terminal domain, 2, RGCC - regulator of cell cycle, GREM1 - gremlin 1, dan family bmp antagonist, SFRP2 - secreted frizzled- related protein 2, APOD - apolipoprotein d, TPM1 - tropomyosin 1 (alpha), HMOX1 - heme oxygenase (decycling) 1, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3] GO:0006006 glucose 3.93E−05 7.95E−03 5.51 [PFKFB4 - 6-phosphofructo-2- metabolic kinase/fructose-2,6-biphosphatase 4, PGK1 - process phosphoglycerate kinase 1, APOD - apolipoprotein d, TPI1 - triosephosphate isomerase 1, PFKP - phosphofructokinase, platelet, PGM1 - phosphoglucomutase 1, PGAM1 - phosphoglycerate mutase 1 (brain), TKT - transketolase, ENO2 - enolase 2 (gamma, neuronal)] GO:2000146 negative 1.69E−05 4.40E−03 5.42 [CITED2 - cbp/p300-interacting regulation of transactivator, with glu/asp-rich carboxy- cell motility terminal domain, 2, RGCC - regulator of cell cycle, GREM1 - gremlin 1, dan family bmp antagonist, SFRP2 - secreted frizzled- related protein 2, APOD - apolipoprotein d, TPM1 - tropomyosin 1 (alpha), HMOX1 - heme oxygenase (decycling) 1, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3] GO:0022617 extracellular 3.28E−04 3.12E−02 5.4 [CTSK - cathepsin k, COL23A1 - matrix collagen, type xxiii, alpha 1, COL8A2 - disassembly collagen, type viii, alpha 2, COL4A1 - collagen, type iv, alpha 1, COL18A1 - collagen, type xviii, alpha 1, COL11A1 - collagen, type xi, alpha 1, COL15A1 - collagen, type xv, alpha 1] GO:0051147 regulation of 3.28E−04 3.09E−02 5.4 [ID3 - inhibitor of dna binding 3, dominant muscle cell negative helix-loop-helix protein, GREM1 - differentiation gremlin 1, dan family bmp antagonist, PRICKLE 1 - prickle homolog 1 (drosophila), RCAN1 - regulator of calcineurin 1, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), IGFBP3 - insulin- like growth factor binding protein 3, CYP26B1 - cytochrome p450, family 26, subfamily b, polypeptide 1] GO:0051271 negative 2.19E−05 5.27E−03 5.26 [CITED2 - cbp/p300-interacting regulation of transactivator, with glu/asp-rich carboxy- cellular terminal domain, 2, RGCC - regulator of component cell cycle, GREM1 - gremlin 1, dan family movement bmp antagonist, SFRP2 - secreted frizzled- related protein 2, APOD - apolipoprotein d, TPM1 - tropomyosin 1 (alpha), HMOX1 - heme oxygenase (decycling) 1, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3] GO:0030198 extracellular 6.57E-10 8.37E−06 5.23 [CTSK - cathepsin k, SFRP2 - secreted matrix frizzled-related protein 2, FOXC1 - organization forkhead box c1, PLOD2 - procollagen- lysine, 2-oxoglutarate 5-dioxygenase 2, JAM2 - junctional adhesion molecule 2, COL8A2 - collagen, type viii, alpha 2, COL23A1 - collagen, type xxiii, alpha 1, COL18A1 - collagen, type xviii, alpha 1, MFAP4 - microfibrillar-associated protein 4, GREM1 - gremlin 1, dan family bmp antagonist, LUM - lumican, VIT - vitrin, CCDC80 - coiled-coil domain containing 80, ABI3BP - abi family, member 3 (nesh) binding protein, COL4A1 - collagen, type iv, alpha 1, COL11A1 - collagen, type xi, alpha 1, GAS6 - growth arrest-specific 6, FBLN2 - fibulin 2, COL15A1 - collagen, type xv, alpha 1, TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b, FBLN1 - fibulin 1] GO:0043062 extracellular 6.91E-10 4.40E−06 5.22 [CTSK - cathepsin k, SFRP2 - secreted structure frizzled-related protein 2, FOXC1 - organization forkhead box c1, PLOD2 - procollagen- lysine, 2-oxoglutarate 5-dioxygenase 2, JAM2 - junctional adhesion molecule 2, COL8A2 - collagen, type viii, alpha 2, COL23A1 - collagen, type xxiii, alpha 1, COL18A1 - collagen, type xviii, alpha 1, MFAP4 - microfibrillar-associated protein 4, GREM1 - gremlin 1, dan family bmp antagonist, LUM - lumican, VIT - vitrin, CCDC80 - coiled-coil domain containing 80, ABI3BP - abi family, member 3 (nesh) binding protein, COL4A1 - collagen, type iv, alpha 1, COL11A1 - collagen, type xi, alpha 1, GAS6 - growth arrest-specific 6, FBLN2 - fibulin 2, COL15A1 - collagen, type xv, alpha 1, TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b, FBLN1 - fibulin 1] GO:0031960 response to 1.63E−04 1.92E−02 5.18 [CTGF - connective tissue growth factor, corticosteroid AKR1C3 - aldo-keto reductase family 1, member c3, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, DUSP1 - dual specificity phosphatase 1, SLIT3 - slit homolog 3 (drosophila), ASS1 - argininosuccinate synthase 1, CCL2 - chemokine (c-c motif) ligand 2, AQP1 - aquaporin 1 (colton blood group)] GO:0030178 negative 4.45E−04 3.61E−02 5.13 [APCDD1 - adenomatosis polyposis coli regulation of down-regulated 1, GREM1 - gremlin 1, Wnt signaling dan family bmp antagonist, PRICKLE 1 - pathway prickle homolog 1 (drosophila), SFRP2 - secreted frizzled-related protein 2, DKK3 - dickkopf wnt signaling pathway inhibitor 3, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), AXIN2 - axin 2] GO:0000302 response to 1.80E−04 2.03E−02 5.11 [AKR1C3 - aldo-keto reductase family 1, reactive oxygen member c3, BNIP3 - bcl2/adenovirus e1b species 19 kda interacting protein 3, APOD - apolipoprotein d, TPM1 - tropomyosin 1 (alpha), DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, KLF2 - kruppel-like factor 2 (lung), AQP1 - aquaporin 1 (colton blood group)] GO:0060828 regulation of 2.88E−04 2.84E−02 4.77 [GREM1 - gremlin 1, dan family bmp canonical Wnt antagonist, PRICKLE 1 - prickle homolog signaling 1 (drosophila), SFRP2 - secreted frizzled- pathway related protein 2, DKK3 - dickkopf wnt signaling pathway inhibitor 3, SOX4 - sry (sex determining region y)-box 4, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), WLS - wntless homolog (drosophila), AXIN2 - axin2] GO:0001818 negative 3.44E−04 3.15E−02 4.65 [RGCC - regulator of cell cycle, TNFSF4 - regulation of tumor necrosis factor (ligand) cytokine superfamily, member 4, APOD - production apolipoprotein d, UBE2L6 - ubiquitin- conjugating enzyme e2l 6, HMOX1 - heme oxygenase (decycling) 1, GAS6 - growth arrest-specific 6, KLF2 - kruppel- like factor 2 (lung), PYCARD - pyd and card domain containing] GO:0007584 response to 3.91E−04 3.30E−02 4.56 [AKR1C3 - aldo-keto reductase family 1, nutrient member c3, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, SFRP2 - secreted frizzled-related protein 2, HMOX1 - heme oxygenase (decycling) 1, GAS6 - growth arrest-specific 6, ASS1 - argininosuccinate synthase 1, CCL2 - chemokine (c-c motif) ligand 2, TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b] GO:0045765 regulation of 1.73E−04 1.97E−02 4.55 [RGCC - regulator of cell cycle, THBS4 - angiogenesis thrombospondin 4, SFRP2 - secreted frizzled-related protein 2, PTGIS - prostaglandin i2 (prostacyclin) synthase, HMOX1 - heme oxygenase (decycling) 1, ACVRL1 - activin a receptor type ii-like 1, F3 - coagulation factor iii (thromboplastin, tissue factor), CCL2 - chemokine (c-c motif) ligand 2, AQP1 - aquaporin 1 (colton blood group)] GO:0040013 negative 7.66E−05 1.28E−02 4.54 [CITED2 - cbp/p300-interacting regulation of transactivator, with glu/asp-rich carboxy- locomotion terminal domain, 2, RGCC - regulator of cell cycle, GREM1 - gremlin 1, dan family bmp antagonist, SFRP2 - secreted frizzled- related protein 2, APOD - apolipoprotein d, TPM1 - tropomyosin 1 (alpha), HMOX1 - heme oxygenase (decycling) 1, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3] GO:0019318 hexose metabolic 2.13E−04 2.28E−02 4.42 [PFKFB4 - 6-phosphofructo-2- process kinase/fructose-2,6-biphosphatase 4, PGK1 - phosphoglycerate kinase 1, APOD - apolipoprotein d, TPI1 - triosephosphate isomerase 1, PFKP - phosphofructokinase, platelet, PGM1 - phosphoglucomutase 1, PGAM1 - phosphoglycerate mutase 1 (brain), TKT - transketolase, ENO2 - enolase 2 (gamma, neuronal)] GO:0043281 regulation of 2.41E−04 2.47E−02 4.35 [CTGF - connective tissue growth factor, cysteine-type SFRP2 - secreted frizzled-related protein endopeptidase 2, TNFAIP8 - tumor necrosis factor, activity alpha-induced protein 8, GAS6 - growth involved in arrest-specific 6, F3 - coagulation factor iii apoptotic (thromboplastin, tissue factor), MICAL1 - process microtubule associated monooxygenase, calponin and lim domain containing 1, AQP1 - aquaporin 1 (colton blood group), PYCARD - pyd and card domain containing, IFI6 - interferon, alpha- inducible protein 6] GO:0001933 negative 2.66E−05 5.84E−03 4.28 [ERRFI1 - erbb receptor feedback regulation of inhibitor 1, GREM1 - gremlin 1, dan protein family bmp antagonist, SFRP2 - secreted phosphorylation frizzled-related protein 2, UCHL1 - ubiquitin carboxyl-terminal esterase l1 (ubiquitin thiolesterase), FAM129A - family with sequence similarity 129, member a, RGS4 - regulator of g-protein signaling 4, PPAP2B - phosphatidic acid phosphatase type 2b, DUSP1 - dual specificity phosphatase 1, SMAD6 - smad family member 6, MICAL1 - microtubule associated monooxygenase, calponin and lim domain containing 1, IGFBP3 - insulin-like growth factor binding protein 3, PYCARD - pyd and card domain containing] GO:0030111 regulation of 1.25E−04 1.69E−02 4.28 [APCDD1 - adenomatosis polyposis coli Wnt signaling down-regulated 1, PRICKLE 1 - prickle pathway homolog 1 (drosophila), GREM1 - gremlin 1, dan family bmp antagonist, SFRP2 - secreted frizzled-related protein 2, DKK3 - dickkopf wnt signaling pathway inhibitor 3, PPAP2B - phosphatidic acid phosphatase type 2b, SOX4 - sry (sex determining region y)-box 4, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), WLS - wntless homolog (drosophila), AXIN2 - axin 2] GO:0048545 response to 5.72E−07 4.86E−04 4.26 [CITED2 - cbp/p300-interacting steroid hormone transactivator, with glu/asp-rich carboxy- terminal domain, 2, AKR1C3 - aldo-keto reductase family 1, member c3, GSTM3 - glutathione s-transferase mu 3 (brain), RCAN1 - regulator of calcineurin 1, SLIT3 - slit homolog 3 (drosophila), CCL2 - chemokine (c-c motif) ligand 2, ASS1 - argininosuccinate synthase 1, CTGF - connective tissue growth factor, WFDC1 - wap four-disulfide core domain 1, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, NR2F1 - nuclear receptor subfamily 2, group f, member 1, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, CA9 - carbonic anhydrase ix, AQP1 - aquaporin 1 (colton blood group), TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b] GO:0001101 response to 1.28E−05 3.69E−03 4.26 [AKR1C3 - aldo-keto reductase family 1, acid chemical member c3, MAP7 - microtubule- associated protein 7, AKR1C4 - aldo-keto reductase family 1, member c4, COL18A1 - collagen, type xviii, alpha 1, CCL2 - chemokine (c-c motif) ligand 2, ASS1 - argininosuccinate synthase 1, CYP26B1 - cytochrome p450, family 26, subfamily b, polypeptide 1, CTGF - connective tissue growth factor, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, COL4A1 - collagen, type iv, alpha 1, CD9 - cd9 molecule, DUSP1 - dual specificity phosphatase 1, AQP1 - aquaporin 1 (colton blood group)] GO:1901342 regulation of 3.29E−04 3.08E−02 4.17 [RGCC - regulator of cell cycle, THBS4 - vasculature thrombospondin 4, SFRP2 - secreted development frizzled-related protein 2, PTGIS - prostaglandin i2 (prostacyclin) synthase, HMOX1 - heme oxygenase (decycling) 1, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, F3 - coagulation factor iii (thromboplastin, tissue factor), AQP1 - aquaporin 1 (colton blood group)] GO:0036293 response to 9.09E−05 1.41E−02 4.07 [CITED2 - cbp/p300-interacting decreased transactivator, with glu/asp-rich carboxy- oxygen levels terminal domain, 2, CTGF - connective tissue growth factor, RGCC - regulator of cell cycle, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, PLOD2 - procollagen-lysine, 2-oxoglutarate 5- dioxygenase 2, PTGIS - prostaglandin i2 (prostacyclin) synthase, HMOX1 - heme oxygenase (decycling) 1, CA9 - carbonic anhydrase ix, CCL2 - chemokine (c-c motif) ligand 2, AQP1 - aquaporin 1 (colton blood group)] GO:0070482 response to 1.49E−04 1.85E−02 3.84 [CITED2 - cbp/p300-interacting oxygen levels transactivator, with glu/asp-rich carboxy- terminal domain, 2, CTGF - connective tissue growth factor, RGCC - regulator of cell cycle, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, PLOD2 - procollagen-lysine, 2-oxoglutarate 5- dioxygenase 2, PTGIS - prostaglandin i2 (prostacyclin) synthase, HMOX1 - heme oxygenase (decycling) 1, CA9 - carbonic anhydrase ix, CCL2 - chemokine (c-c motif) ligand 2, AQP1 - aquaporin 1 (colton blood group)] GO:0001525 angiogenesis 3.11E−04 3.03E−02 3.82 [CTGF - connective tissue growth factor, HAND2 - heart and neural crest derivatives expressed 2, APOD - apolipoprotein d, COL8A2 - collagen, type viii, alpha 2, COL18A1 - collagen, type xviii, alpha 1, HMOX1 - heme oxygenase (decycling) 1, ACVRL1 - activin a receptor type ii-like 1, ANPEP - alanyl (membrane) aminopeptidase, CCL2 - chemokine (c-c motif) ligand 2, COL15A1 - collagen, type xv, alpha 1] GO:0001666 response to 3.56E−04 3.20E−02 3.76 [CITED2 - cbp/p300-interacting hypoxia transactivator, with glu/asp-rich carboxy- terminal domain, 2, RGCC - regulator of cell cycle, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, PLOD2 - procollagen-lysine, 2-oxoglutarate 5- dioxygenase 2, PTGIS - prostaglandin i2 (prostacyclin) synthase, HMOX1 - heme oxygenase (decycling) 1, CA9 - carbonic anhydrase ix, CCL2 - chemokine (c-c motif) ligand 2, AQP1 - aquaporin 1 (colton blood group)] GO:0030334 regulation of 2.53E−07 2.69E−04 3.71 [CITED2 - cbp/p300-interacting cell migration transactivator, with glu/asp-rich carboxy- terminal domain, 2, SFRP2 - secreted frizzled-related protein 2, TNFRSF14 - tumor necrosis factor receptor superfamily, member 14, TPM1 - tropomyosin 1 (alpha), COL18A1 - collagen, type xviii, alpha 1, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, BMPER - bmp binding endothelial regulator, RGCC - regulator of cell cycle, GREM1 - gremlin 1, dan family bmp antagonist, THBS4 - thrombospondin 4, CXCL16 - chemokine (c-x-c motif) ligand 16, ENPP2 - ectonucleotide pyrophosphatase/phosphodiesterase 2, APOD - apolipoprotein d, HMOX1 - heme oxygenase (decycling) 1, GAS6 - growth arrest-specific 6, F3 - coagulation factor iii (thromboplastin, tissue factor), AQP1 - aquaporin 1 (colton blood group), PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3] GO:0060429 epithelium 5.58E−04 4.25E−02 3.55 [ERRFI1 - erbb receptor feedback development inhibitor 1, CTGF - connective tissue growth factor, APCDD1 - adenomatosis polyposis coli down-regulated 1, FOXC1 - forkhead box c1, ALDH3A2 - aldehyde dehydrogenase 3 family, member a2, TFAP2C - transcription factor ap-2 gamma (activating enhancer binding protein 2 gamma), KRT34 - keratin 34, SMAD6 - smad family member 6, AQP1 - aquaporin 1 (colton blood group), BMPER - bmp binding endothelial regulator] GO:0042326 negative 1.56E−04 1.88E−02 3.55 [ERRFI1 - erbb receptor feedback regulation of inhibitor 1, GREM1 - gremlin 1, dan phosphorylation family bmp antagonist, SFRP2 - secreted frizzled-related protein 2, UCHL1 - ubiquitin carboxyl-terminal esterase l1 (ubiquitin thiolesterase), FAM129A - family with sequence similarity 129, member a, RGS4 - regulator of g-protein signaling 4, PPAP2B - phosphatidic acid phosphatase type 2b, DUSP1 - dual specificity phosphatase 1, SMAD6 - smad family member 6, MICAL1 - microtubule associated monooxygenase, calponin and lim domain containing 1, IGFBP3 - insulin-like growth factor binding protein 3, PYCARD - pyd and card domain containing] GO:2000145 regulation of 5.93E−07 4.72E−04 3.52 [CITED2 - cbp/p300-interacting cell motility transactivator, with glu/asp-rich carboxy- terminal domain, 2, SFRP2 - secreted frizzled-related protein 2, TNFRSF14 - tumor necrosis factor receptor superfamily, member 14, TPM1 - tropomyosin 1 (alpha), COL18A1 - collagen, type xviii, alpha 1, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, BMPER - bmp binding endothelial regulator, RGCC - regulator of cell cycle, GREM1 - gremlin 1, dan family bmp antagonist, THBS4 - thrombospondin 4, CXCL16 - chemokine (c-x-c motif) ligand 16, ENPP2 - ectonucleotide pyrophosphatase/phosphodiesterase 2, APOD - apolipoprotein d, HMOX1 - heme oxygenase (decycling) 1, GAS6 - growth arrest-specific 6, F3 - coagulation factor iii (thromboplastin, tissue factor), AQP1 - aquaporin 1 (colton blood group), PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3] GO:0030155 regulation of 9.07E−05 1.43E−02 3.52 [CITED2 - cbp/p300-interacting cell adhesion transactivator, with glu/asp-rich carboxy- terminal domain, 2, SFRP2 - secreted frizzled-related protein 2, JAM2 - junctional adhesion molecule 2, TPM1 - tropomyosin 1 (alpha), ACVRL1 - activin a receptor type ii-like 1, ASS1 - argininosuccinate synthase 1, RGCC - regulator of cell cycle, APOD - apolipoprotein d, VIT - vitrin, CCDC80 - coiled-coil domain containing 80, ABI3BP - abi family, member 3 (nesh) binding protein, NUAK1 - nuak family, snf1-like kinase, 1, FBLN2 - fibulin 2] GO:0051270 regulation of 1.94E−06 9.90E−04 3.27 [CITED2 - cbp/p300-interacting cellular transactivator, with glu/asp-rich carboxy- component terminal domain, 2, SFRP2 - secreted movement frizzled-related protein 2, TNFRSF14 - tumor necrosis factor receptor superfamily, member 14, TPM1 - tropomyosin 1 (alpha), COL18A1 - collagen, type xviii, alpha 1, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, BMPER - bmp binding endothelial regulator, RGCC - regulator of cell cycle, GREM1 - gremlin 1, dan family bmp antagonist, THBS4 - thrombospondin 4, CXCL16 - chemokine (c-x-c motif) ligand 16, ENPP2 - ectonucleotide pyrophosphatase/phosphodiesterase 2, APOD - apolipoprotein d, HMOX1 - heme oxygenase (decycling) 1, GAS6 - growth arrest-specific 6, F3 - coagulation factor iii (thromboplastin, tissue factor), AQP1 - aquaporin 1 (colton blood group), PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3] GO:0010942 positive 6.57E−05 1.16E−02 3.25 [AKR1C3 - aldo-keto reductase family 1, regulation of member c3, ID3 - inhibitor of dna binding cell death 3, dominant negative helix-loop-helix protein, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, SFRP2 - secreted frizzled-related protein 2, PAWR - prkc, apoptosis, wt1, regulator, ARHGEF3 - rho guanine nucleotide exchange factor (gef) 3, COL18A1 - collagen, type xviii, alpha 1, SOX4 - sry (sex determining region y)-box 4, IGFBP3 - insulin-like growth factor binding protein 3, CTGF - connective tissue growth factor, RGCC - regulator of cell cycle, DUSP1 - dual specificity phosphatase 1, AXIN2 - axin 2, PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3] GO:0010035 response to 2.26E−04 2.36E−02 3.21 [AKR1C3 - aldo-keto reductase family 1, inorganic member c3, BNIP3 - bcl2/adenovirus e1b substance 19 kda interacting protein 3, COL18A1 - collagen, type xviii, alpha 1, KLF2 - kruppel-like factor 2 (lung), ASS1 - argininosuccinate synthase 1, MT1M - metallothionein 1m, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, CD9 - cd9 molecule, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, AQP1 - aquaporin 1 (colton blood group), CYBRD1- cytochrome b reductase 1, TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b] GO:0040012 regulation of 2.76E−06 1.26E−03 3.2 [CITED2 - cbp/p300-interacting locomotion transactivator, with glu/asp-rich carboxy- terminal domain, 2, SFRP2 - secreted frizzled-related protein 2, TNFRSF14 - tumor necrosis factor receptor superfamily, member 14, TPM1 - tropomyosin 1 (alpha), COL18A1 - collagen, type xviii, alpha 1, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, BMPER - bmp binding endothelial regulator, RGCC - regulator of cell cycle, GREM1 - gremlin 1, dan family bmp antagonist, THBS4 - thrombospondin 4, CXCL16 - chemokine (c-x-c motif) ligand 16, ENPP2 - ectonucleotide pyrophosphatase/phosphodiesterase 2, APOD - apolipoprotein d, HMOX1 - heme oxygenase (decycling) 1, GAS6 - growth arrest-specific 6, F3 - coagulation factor iii (thromboplastin, tissue factor), AQP1 - aquaporin 1 (colton blood group), PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3] GO:0001817 regulation of 9.31E−05 1.43E−02 3.01 [UBE2L6 - ubiquitin-conjugating enzyme cytokine e2l 6, TNFRSF14 - tumor necrosis factor production receptor superfamily, member 14, HLA- DPA1 - major histocompatibility complex, class ii, dp alpha 1, NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, SRGN - serglycin, CCL2 - chemokine (c-c motif) ligand 2, KLF2 - kruppel-like factor 2 (lung), CARD9 - caspase recruitment domain family, member 9, RGCC - regulator of cell cycle, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, ADORA2B - adenosine a2b receptor, APOD - apolipoprotein d, HMOX1 - heme oxygenase (decycling) 1, GAS6 - growth arrest-specific 6, PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3] GO:0043068 positive 4.29E−04 3.52E−02 3 [AKR1C3 - aldo-keto reductase family 1, regulation of member c3, ID3 - inhibitor of dna binding programmed 3, dominant negative helix-loop-helix cell death protein, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, SFRP2 - secreted frizzled-related protein 2, PAWR - prkc, apoptosis, wt1, regulator, ARHGEF3 - rho guanine nucleotide exchange factor (gef) 3, COL18A1 - collagen, type xviii, alpha 1, SOX4 - sry (sex determining region y)-box 4, IGFBP3 - insulin-like growth factor binding protein 3, RGCC - regulator of cell cycle, DUSP1 - dual specificity phosphatase 1, PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3] GO:0008283 cell 6.05E−05 1.09E−02 2.77 [CITED2 - cbp/p300-interacting proliferation transactivator, with glu/asp-rich carboxy- terminal domain, 2, MAP7 - microtubule- associated protein 7, FOXC1 - forkhead box c1, SFRP2 - secreted frizzled-related protein 2, UCHL1 - ubiquitin carboxyl- terminal esterase l1 (ubiquitin thiolesterase), COL8A2 - collagen, type viii, alpha 2, SOX4 - sry (sex determining region y)-box 4, ACVRL1 - activin a receptor type ii-like 1, IGFBP3 - insulin-like growth factor binding protein 3, BMPER - bmp binding endothelial regulator, CTGF - connective tissue growth factor, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, HAND2 - heart and neural crest derivatives expressed 2, TFAP2C - transcription factor ap-2 gamma (activating enhancer binding protein 2 gamma), HMOX1 - heme oxygenase (decycling) 1, OSR2 - odd-skipped related 2 (drosophila), GAS6 - growth arrest- specific 6, AXIN2 - axin2, BCAT1 - branched chain amino-acid transaminase 1, cytosolic] GO:0008285 negative 1.14E−04 1.59E−02 2.73 [SFRP2 - secreted frizzled-related protein regulation of 2, ADAMTS1 - adam metallopeptidase cell with thrombospondin type 1 motif, 1, proliferation PAWR - prkc, apoptosis, wt1, regulator, TNFRSF14 - tumor necrosis factor receptor superfamily, member 14, COL18A1 - collagen, type xviii, alpha 1, SLIT3 - slit homolog 3 (drosophila), SOX4 - sry (sex determining region y)-box 4, ACVRL1 - activin a receptor type ii-like 1, IGFBP3 - insulin-like growth factor binding protein 3, WFDC1 - wap four- disulfide core domain 1, RGCC - regulator of cell cycle, GREM1 - gremlin 1, dan family bmp antagonist, APOD - apolipoprotein d, CD9 - cd9 molecule, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, AXIN2 - axin 2, GAS1 - growth arrest-specific 1] GO:0048468 cell 6.35E−04 4.70E−02 2.63 [CITED2 - cbp/p300-interacting development transactivator, with glu/asp-rich carboxy- terminal domain, 2, MAP7 - microtubule- associated protein 7, SFRP2 - secreted frizzled-related protein 2, FOXC1 - forkhead box c1, UCHL1 - ubiquitin carboxyl-terminal esterase l1 (ubiquitin thiolesterase), GSTM3 - glutathione s- transferase mu 3 (brain), SHROOM3 - shroom family member 3, RCAN1 - regulator of calcineurin 1, SOX4 - sry (sex determining region y)-box 4, HAND2 - heart and neural crest derivatives expressed 2, TFAP2C - transcription factor ap-2 gamma (activating enhancer binding protein 2 gamma), CD9 - cd9 molecule, HSPA2 - heat shock 70 kda protein 2, COL11A1 - collagen, type xi, alpha 1, FHL2 - four and a half lim domains 2] GO:0008284 positive 9.52E−05 1.44E−02 2.52 [AKR1C3 - aldo-keto reductase family 1, regulation of member c3, SFRP2 - secreted frizzled- cell related protein 2, COL18A1 - collagen, proliferation type xviii, alpha 1, HLA-DPA1 - major histocompatibility complex, class ii, dp alpha 1, SOX4 - sry (sex determining region y)-box 4, ACVRL1 - activin a receptor type ii-like 1, CCL2 - chemokine (c-c motif) ligand 2, CTGF - connective tissue growth factor, GREM1 - gremlin 1, dan family bmp antagonist, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, THBS4 - thrombospondin 4, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, HMOX1 - heme oxygenase (decycling) 1, OSR2 - odd- skipped related 2 (drosophila), GAS6 - growth arrest-specific 6, TNS3 - tensin 3, F3 - coagulation factor iii (thromboplastin, tissue factor), MARCKSL1 - marcks-like 1, GAS1 - growth arrest-specific 1, AQP1 - aquaporin 1 (colton blood group), PYCARD - pyd and card domain containing] GO:0016477 cell migration 3.85E−04 3.27E−02 2.47 [SORBS2 - sorbin and sh3 domain containing 2, APCDD1 - adenomatosis polyposis coli down-regulated 1, FOXC1 - forkhead box c1, JAM2 - junctional adhesion molecule 2, ACVRL1 - activin a receptor type ii-like 1, PROS 1 - protein s (alpha), CDH2 - cadherin 2, type 1, n- cadherin (neuronal), CCL2 - chemokine (c-c motif) ligand 2, CENPV - centromere protein v, CTGF - connective tissue growth factor, GREM1 - gremlin 1, dan family bmp antagonist, THBS4 - thrombospondin 4, CXCL16 - chemokine (c-x-c motif) ligand 16, HAND2 - heart and neural crest derivatives expressed 2, NR2F1 - nuclear receptor subfamily 2, group f, member 1, PPAP2B - phosphatidic acid phosphatase type 2b, GAS6 - growth arrest-specific 6, TNS3 - tensin 3] GO:0042127 regulation of 5.13E−07 4.67E−04 2.44 [AKR1C3 - aldo-keto reductase family 1, cell member c3, ADAMTS1 - adam proliferation metallopeptidase with thrombospondin type 1 motif, 1, PAWR - prkc, apoptosis, wt1, regulator, COL18A1 - collagen, type xviii, alpha 1, ACVRL1 - activin a receptor type ii-like 1, CTGF - connective tissue growth factor, RGCC - regulator of cell cycle, WFDC1 - wap four-disulfide core domain 1, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, HAND2 - heart and neural crest derivatives expressed 2, CD9 - cd9 molecule, TNS3 - tensin 3, AXIN2 - axin 2, PYCARD - pyd and card domain containing, RIPK3 - receptor- interacting serine-threonine kinase 3, SFRP2 - secreted frizzled-related protein 2, TNFRSF14 - tumor necrosis factor receptor superfamily, member 14, HLA- DPA1 - major histocompatibility complex, class ii, dp alpha 1, SLIT3 - slit homolog 3 (drosophila), NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b- cells inhibitor, alpha, SOX4 - sry (sex determining region y)-box 4, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, GREM1 - gremlin 1, dan family bmp antagonist, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, THBS4 - thrombospondin 4, APOD - apolipoprotein d, NUAK1 - nuak family, snf1-like kinase, 1, OSR2 - odd-skipped related 2 (drosophila), HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, GAS6 - growth arrest- specific 6, F3 - coagulation factor iii (thromboplastin, tissue factor), MARCKSL1 - marcks-like 1, GAS1 - growth arrest-specific 1, AQP1 - aquaporin 1 (colton blood group)] GO:0023057 negative 8.06E−05 1.30E−02 2.36 [ERRFI1 - erbb receptor feedback regulation of inhibitor 1, APCDD1 - adenomatosis signaling polyposis coli down-regulated 1, SFRP2 - secreted frizzled-related protein 2, UCHL1 - ubiquitin carboxyl-terminal esterase l1 (ubiquitin thiolesterase), PAWR - prkc, apoptosis, wt1, regulator, SLIT3 - slit homolog 3 (drosophila), NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), CYP26B1 - cytochrome p450, family 26, subfamily b, polypeptide 1, IGFBP3 - insulin-like growth factor binding protein 3, BMPER - bmp binding endothelial regulator, PRICKLE 1 - prickle homolog 1 (drosophila), GREM1 - gremlin 1, dan family bmp antagonist, DKK3 - dickkopf wnt signaling pathway inhibitor 3, APOD - apolipoprotein d, RGS4 - regulator of g-protein signaling 4, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, GAS6 - growth arrest-specific 6, AXIN2 - axin 2, GAS1 - growth arrest-specific 1, PYCARD - pyd and card domain containing, IFI6 - interferon, alpha-inducible protein 6] GO:0010648 negative 8.34E−05 1.33E−02 2.36 [ERRFI1 - erbb receptor feedback regulation of inhibitor 1, APCDD1 - adenomatosis cell polyposis coli down-regulated 1, SFRP2 - communication secreted frizzled-related protein 2, UCHL1 - ubiquitin carboxyl-terminal esterase l1 (ubiquitin thiolesterase), PAWR - prkc, apoptosis, wt1, regulator, SLIT3 - slit homolog 3 (drosophila), NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), CYP26B1 - cytochrome p450, family 26, subfamily b, polypeptide 1, IGFBP3 - insulin-like growth factor binding protein 3, BMPER - bmp binding endothelial regulator, PRICKLE 1 - prickle homolog 1 (drosophila), GREM1 - gremlin 1, dan family bmp antagonist, DKK3 - dickkopf wnt signaling pathway inhibitor 3, APOD - apolipoprotein d, RGS4 - regulator of g-protein signaling 4, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, GAS6 - growth arrest-specific 6, AXIN2 - axin 2, GAS1 - growth arrest-specific 1, PYCARD - pyd and card domain containing, IFI6 - interferon, alpha-inducible protein 6] GO:0060548 negative 4.72E−04 3.73E−02 2.36 [CITED2 - cbp/p300-interacting regulation of transactivator, with glu/asp-rich carboxy- cell death terminal domain, 2, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, FOXC1 - forkhead box c1, SFRP2 - secreted frizzled-related protein 2, EGR2 - early growth response 2, NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, SOX4 - sry (sex determining region y)-box 4, MICAL1 - microtubule associated monooxygenase, calponin and lim domain containing 1, CCL2 - chemokine (c-c motif) ligand 2, CTGF - connective tissue growth factor, HAND2 - heart and neural crest derivatives expressed 2, TNFAIP8 - tumor necrosis factor, alpha-induced protein 8, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, GAS6 - growth arrest-specific 6, FHL2 - four and a half lim domains 2, GAS1 - growth arrest- specific 1, AQP1 - aquaporin 1 (colton blood group)] GO:0009605 response to 3.86E−06 1.59E−03 2.3 [CITED2 - cbp/p300-interacting external transactivator, with glu/asp-rich carboxy- stimulus terminal domain, 2, AKR1C3 - aldo-keto reductase family 1, member c3, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, SHROOM3 - shroom family member 3, BAIAP2L1 - bail-associated protein 2-like 1, RCAN1 - regulator of calcineurin 1, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, ENPP2 - ectonucleotide pyrophosphatase/phosphodiesterase 2, CXCL16 - chemokine (c-x-c motif) ligand 16, MX1 - myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse), COTL1 - coactosin-like 1 (dictyostelium), OAS2 - 2′-5′- oligoadenylate synthetase 2, 69/71 kda, PYCARD - pyd and card domain containing, BHLHE40 - basic helix-loop- helix family, member e40, SFRP2 - secreted frizzled-related protein 2, AKR1C4 - aldo-keto reductase family 1, member c4, TNFRSF14 - tumor necrosis factor receptor superfamily, member 14, PENK - proenkephalin, SLIT3 - slit homolog 3 (drosophila), NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b- cells inhibitor, alpha, ASS1 - argininosuccinate synthase 1, CCL2 - chemokine (c-c motif) ligand 2, CARD9 - caspase recruitment domain family, member 9, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, ADORA2B - adenosine a2b receptor, RDH5 - retinol dehydrogenase 5 (11-cis/9-cis), TNFAIP8 - tumor necrosis factor, alpha-induced protein 8, HMOX1 - heme oxygenase (decycling) 1, IFI44 - interferon-induced protein 44, COL11A1 - collagen, type xi, alpha 1, GAS6 - growth arrest-specific 6, ANPEP - alanyl (membrane) aminopeptidase, AQP1 - aquaporin 1 (colton blood group), TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b] GO:1901700 response to 2.31E−05 5.36E−03 2.3 [AKR1C3 - aldo-keto reductase family 1, oxygen- member c3, BNIP3 - bcl2/adenovirus e1b containing 19 kda interacting protein 3, MAP7 - compound microtubule-associated protein 7, TPM1 - tropomyosin 1 (alpha), COL18A1 - collagen, type xviii, alpha 1, KLF2 - kruppel-like factor 2 (lung), CYP26B1 - cytochrome p450, family 26, subfamily b, polypeptide 1, CTGF - connective tissue growth factor, WFDC1 - wap four- disulfide core domain 1, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, CD9 - cd9 molecule, COL4A1 - collagen, type iv, alpha 1, CA9 - carbonic anhydrase ix, PYCARD - pyd and card domain containing, AKR1C4 - aldo-keto reductase family 1, member c4, SLIT3 - slit homolog 3 (drosophila), EGR2 - early growth response 2, NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, SOX4 - sry (sex determining region y)-box 4, ASS1 - argininosuccinate synthase 1, CCL2 - chemokine (c-c motif) ligand 2, CARD9 - caspase recruitment domain family, member 9, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, APOD - apolipoprotein d, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, GAS6 - growth arrest-specific 6, AQP1 - aquaporin 1 (colton blood group), ADCY4 - adenylate cyclase 4] GO:0040011 locomotion 3.52E−04 3.18E−02 2.29 [APCDD1 - adenomatosis polyposis coli down-regulated 1, SORBS2 - sorbin and sh3 domain containing 2, FOXC1 - forkhead box c1, JAM2 - junctional adhesion molecule 2, RCAN1 - regulator of calcineurin 1, SLIT3 - slit homolog 3 (drosophila), ACVRL1 - activin a receptor type ii-like 1, PROS1 - protein s (alpha), CDH2 - cadherin 2, type 1, n-cadherin (neuronal), CCL2 - chemokine (c-c motif) ligand 2, CENPV - centromere protein v, CTGF - connective tissue growth factor, GREM1 - gremlin 1, dan family bmp antagonist, THBS4 - thrombospondin 4, HAND2 - heart and neural crest derivatives expressed 2, ENPP2 - ectonucleotide pyrophosphatase/phosphodiesterase 2, CXCL16 - chemokine (c-x-c motif) ligand 16, NR2F1 - nuclear receptor subfamily 2, group f, member 1, PPAP2B - phosphatidic acid phosphatase type 2b, GAS6 - growth arrest-specific 6, TNS3 - tensin 3] GO:0042981 regulation of 1.12E−05 3.49E−03 2.26 [CITED2 - cbp/p300-interacting apoptotic transactivator, with glu/asp-rich carboxy- process terminal domain, 2, AKR1C3 - aldo-keto reductase family 1, member c3, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, PAWR - prkc, apoptosis, wt1, regulator, COL18A1 - collagen, type xviii, alpha 1, MICAL1 - microtubule associated monooxygenase, calponin and lim domain containing 1, CTGF - connective tissue growth factor, RGCC - regulator of cell cycle, HAND2 - heart and neural crest derivatives expressed 2, PTGIS - prostaglandin i2 (prostacyclin) synthase, PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3, ID3 - inhibitor of dna binding 3, dominant negative helix- loop-helix protein, FOXC1 - forkhead box c1, SFRP2 - secreted frizzled-related protein 2, ARHGEF3 - rho guanine nucleotide exchange factor (gef) 3, EGR2 - early growth response 2, NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, SOX4 - sry (sex determining region y)-box 4, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, CARD9 - caspase recruitment domain family, member 9, TNFAIP8 - tumor necrosis factor, alpha- induced protein 8, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, GAS6 - growth arrest- specific 6, FHL2 - four and a half lim domains 2, F3 - coagulation factor iii (thromboplastin, tissue factor), GAS1 - growth arrest-specific 1, AQP1 - aquaporin 1 (colton blood group), IFI6 - interferon, alpha-inducible protein 6] GO:0009719 response to 1.61E−05 4.28E−03 2.26 [CITED2 - cbp/p300-interacting endogenous transactivator, with glu/asp-rich carboxy- stimulus terminal domain, 2, GSTM2 - glutathione s-transferase mu 2 (muscle), AKR1C3 - aldo-keto reductase family 1, member c3, GSTM3 - glutathione s-transferase mu 3 (brain), RCAN1 - regulator of calcineurin 1, ACVRL1 - activin a receptor type ii-like 1, EEF1A1 - eukaryotic translation elongation factor 1 alpha 1, KLF2 - kruppel-like factor 2 (lung), CTGF - connective tissue growth factor, WFDC1 - wap four-disulfide core domain 1, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, NR2F1 - nuclear receptor subfamily 2, group f, member 1, COL4A1 - collagen, type iv, alpha 1, CA9 - carbonic anhydrase ix, AKR1C4 - aldo- keto reductase family 1, member c4, SLIT3 - slit homolog 3 (drosophila), EGR2 - early growth response 2, NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, ASS1 - argininosuccinate synthase 1, CCL2 - chemokine (c-c motif) ligand 2, CARD9 - caspase recruitment domain family, member 9, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, CLEC3B - c-type lectin domain family 3, member b, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, FHL2 - four and a half lim domains 2, AQP1 - aquaporin 1 (colton blood group), TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b, ADCY4 - adenylate cyclase 4, STMN2 - stathmin-like 2] GO:0007155 cell adhesion 4.13E−04 3.44E−02 2.26 [SORBS2 - sorbin and sh3 domain containing 2, JAM2 - junctional adhesion molecule 2, COL8A2 - collagen, type viii, alpha 2, COL18A1 - collagen, type xviii, alpha 1, ISLR - immunoglobulin superfamily containing leucine-rich repeat, ANTXR1 - anthrax toxin receptor 1, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), CCL2 - chemokine (c-c motif) ligand 2, MFAP4 - microfibrillar- associated protein 4, CTGF - connective tissue growth factor, THBS4 - thrombospondin 4, CLDN11 - claudin 11, CD9 - cd9 molecule, PPAP2B - phosphatidic acid phosphatase type 2b, NUAK1 - nuak family, snf1-like kinase, 1, CSRP1 - cysteine and glycine-rich protein 1, SMAD6 - smad family member 6, COL11A1 - collagen, type xi, alpha 1, GAS6 - growth arrest-specific 6, ALCAM - activated leukocyte cell adhesion molecule, COL15A1 - collagen, type xv, alpha 1] GO:0022610 biological 4.33E−04 3.54E−02 2.25 [SORBS2 - sorbin and sh3 domain adhesion containing 2, JAM2 - junctional adhesion molecule 2, COL8A2 - collagen, type viii, alpha 2, COL18A1 - collagen, type xviii, alpha 1, ISLR - immunoglobulin superfamily containing leucine-rich repeat, ANTXR1 - anthrax toxin receptor 1, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), CCL2 - chemokine (c-c motif) ligand 2, MFAP4 - microfibrillar- associated protein 4, CTGF - connective tissue growth factor, THBS4 - thrombospondin 4, CLDN11 - claudin 11, CD9 - cd9 molecule, PPAP2B - phosphatidic acid phosphatase type 2b, NUAK1 - nuak family, snf1-like kinase, 1, CSRP1 - cysteine and glycine-rich protein 1, SMAD6 - smad family member 6, COL11A1 - collagen, type xi, alpha 1, GAS6 - growth arrest-specific 6, ALCAM - activated leukocyte cell adhesion molecule, COL15A1 - collagen, type xv, alpha 1] GO:0043067 regulation of 1.32E−05 3.65E−03 2.24 [CITED2 - cbp/p300-interacting programmed transactivator, with glu/asp-rich carboxy- cell death terminal domain, 2, AKR1C3 - aldo-keto reductase family 1, member c3, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, PAWR - prkc, apoptosis, wt1, regulator, COL18A1 - collagen, type xviii, alpha 1, MICAL1 - microtubule associated monooxygenase, calponin and lim domain containing 1, CTGF - connective tissue growth factor, RGCC - regulator of cell cycle, HAND2 - heart and neural crest derivatives expressed 2, PTGIS - prostaglandin i2 (prostacyclin) synthase, PYCARD - pyd and card domain containing, RIPK3 - receptor-interacting serine-threonine kinase 3, ID3 - inhibitor of dna binding 3, dominant negative helix- loop-helix protein, FOXC1 - forkhead box c1, SFRP2 - secreted frizzled-related protein 2, ARHGEF3 - rho guanine nucleotide exchange factor (gef) 3, EGR2 - early growth response 2, NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, SOX4 - sry (sex determining region y)-box 4, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, CARD9 - caspase recruitment domain family, member 9, TNFAIP8 - tumor necrosis factor, alpha- induced protein 8, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, GAS6 - growth arrest- specific 6, FHL2 - four and a half lim domains 2, F3 - coagulation factor iii (thromboplastin, tissue factor), GAS1 - growth arrest-specific 1, AQP1 - aquaporin 1 (colton blood group), IFI6 - interferon, alpha-inducible protein 6] GO:0010941 regulation of 1.13E−05 3.41E−03 2.23 [CITED2 - cbp/p300-interacting cell death transactivator, with glu/asp-rich carboxy- terminal domain, 2, AKR1C3 - aldo-keto reductase family 1, member c3, BNIP3 - bcl2/adenovirus e1b 19 kda interacting protein 3, PAWR - prkc, apoptosis, wt1, regulator, COL18A1 - collagen, type xviii, alpha 1, MICAL1 - microtubule associated monooxygenase, calponin and lim domain containing 1, CTGF - connective tissue growth factor, RGCC - regulator of cell cycle, HAND2 - heart and neural crest derivatives expressed 2, PTGIS - prostaglandin i2 (prostacyclin) synthase, AXIN2 - axin 2, PYCARD - pyd and card domain containing, RIPK3 - receptor- interacting serine-threonine kinase 3, ID3 - inhibitor of dna binding 3, dominant negative helix-loop-helix protein, FOXC1 - forkhead box c1, SFRP2 - secreted frizzled-related protein 2, ARHGEF3 - rho guanine nucleotide exchange factor (gef) 3, EGR2 - early growth response 2, NFKBIA - nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor, alpha, SOX4 - sry (sex determining region y)-box 4, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, CARD9 - caspase recruitment domain family, member 9, TNFAIP8 - tumor necrosis factor, alpha- induced protein 8, DUSP1 - dual specificity phosphatase 1, HMOX1 - heme oxygenase (decycling) 1, SMAD6 - smad family member 6, GAS6 - growth arrest- specific 6, FHL2 - four and a half lim domains 2, F3 - coagulation factor iii (thromboplastin, tissue factor), GAS1 - growth arrest-specific 1, AQP1 - aquaporin 1 (colton blood group), IFI6 - interferon, alpha-inducible protein 6]

Molecular Function

GO Term Description P-value FDR q-value Enrichment Genes GO:0001968 fibronectin binding 7.36E−06 3.00E−02 17.89 CTGF, CTSK, SFRP2, CCDC80, IGFBP3 GO:0005201 extracellular matrix 1.07E−05 2.18E−02 9.21 LUM, COL8A2, COL4A1, structural constituent COL11A1, FBLN2, COL15A1, FBLN1

Cellular Component

GO Term Description P-value FDR q-value Enrichment Genes GO:0005604 basement 1.57E−05 2.29E−03 8.7 [THBS4 - thrombospondin 4, ADAMTS1 - membrane adam metallopeptidase with thrombospondin type 1 motif, 1, COL8A2 - collagen, type viii, alpha 2, CCDC80 - coiled-coil domain containing 80, COL4A1 - collagen, type iv, alpha 1, COL18A1 - collagen, type xviii, alpha 1, FBLN1 - fibulin 1] GO:0044420 extracellular 1.34E−07 2.45E−05 8 [THBS4 - thrombospondin 4, LUM - matrix part lumican, ADAMTS1 - adam metallopeptidase with thrombospondin type 1 motif, 1, COL8A2 - collagen, type viii, alpha 2, CCDC80 - coiled-coil domain containing 80, COL4A1 - collagen, type iv, alpha 1, COL18A1 - collagen, type xviii, alpha 1, COL11A1 - collagen, type xi, alpha 1, COL15A1 - collagen, type xv, alpha 1, FBLN1 - fibulin 1, MFAP4 - microfibrillar- associated protein 4] GO:0005581 collagen 5.80E−05 7.73E−03 7.11 [LUM - lumican, COL8A2 - collagen, trimer type viii, alpha 2, COL23A1 - collagen, type xxiii, alpha 1, COL4A1 - collagen, type iv, alpha 1, COL18A1 - collagen, type xviii, alpha 1, COL11A1 - collagen, type xi, alpha 1, COL15A1 - collagen, type xv, alpha 1] GO:0031012 extracellular 2.66E−07 4.33E−05 4.5 [SFRP2 - secreted frizzled-related protein matrix 2, COL8A2 - collagen, type viii, alpha 2, COL18A1 - collagen, type xviii, alpha 1, MFAP4 - microfibrillar-associated protein 4, CTGF - connective tissue growth factor, THBS4 - thrombospondin 4, LUM - lumican, CLEC3B - c-type lectin domain family 3, member b, VIT - vitrin, CCDC80 - coiled-coil domain containing 80, COL4A1 - collagen, type iv, alpha 1, ABI3BP - abi family, member 3 (nesh) binding protein, F3 - coagulation factor iii (thromboplastin, tissue factor), FBLN2 - fibulin2, COL15A1 - collagen, type xv, alpha 1, FBLN1 - fibulin 1, TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b] GO:0005788 endoplasmic 2.04E−04 2.31E−02 4.45 [RDH5 - retinol dehydrogenase 5 (11- reticulum cis/9-cis), GPX7 - glutathione peroxidase lumen 7, COL23A1 - collagen, type xxiii, alpha 1, COL8A2 - collagen, type viii, alpha 2, COL4A1 - collagen, type iv, alpha 1, COL18A1 - collagen, type xviii, alpha 1, COL11A1 - collagen, type xi, alpha 1, GAS6 - growth arrest-specific 6, COL15A1 - collagen, type xv, alpha 1] GO:0005615 extracellular 3.94E−10 2.89E−07 3.09 [LOXL4 - lysyl oxidase-like 4, TPI1 - space triosephosphate isomerase 1, KRT34 - keratin 34, COL18A1 - collagen, type xviii, alpha 1, EEF1A1 - eukaryotic translation elongation factor 1 alpha 1, PROS1 - protein s (alpha), SRGN - serglycin, CTGF - connective tissue growth factor, WFDC1 - wap four- disulfide core domain 1, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, LUM - lumican, ENPP2 - ectonucleotide pyrophosphatase/phosphodiesterase 2, CXCL16 - chemokine (c-x-c motif) ligand 16, DKK3 - dickkopf wnt signaling pathway inhibitor 3, PTGIS - prostaglandin i2 (prostacyclin) synthase, CD9 - cd9 molecule, ENO2 - enolase 2 (gamma, neuronal), FBLN1 - fibulin 1, CTSK - cathepsin k, SFRP2 - secreted frizzled-related protein 2, CFB - complement factor b, SLIT3 - slit homolog 3 (drosophila), CTSZ - cathepsin z, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, BMPER - bmp binding endothelial regulator, GREM1 - gremlin 1, dan family bmp antagonist, ALDH3A1 - aldehyde dehydrogenase 3 family, member a1, THBS4 - thrombospondin 4, CLEC3B - c-type lectin domain family 3, member b, APOD - apolipoprotein d, ABI3BP - abi family, member 3 (nesh) binding protein, HMOX1 - heme oxygenase (decycling) 1, ANPEP - alanyl (membrane) aminopeptidase, GAS6 - growth arrest- specific 6, F3 - coagulation factor iii (thromboplastin, tissue factor), COL15A1 - collagen, type xv, alpha 1, TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b] GO:0044432 endoplasmic 3.38E−04 3.53E−02 2.15 [LSS - lanosterol synthase (2,3- reticulum oxidosqualene-lanosterol cyclase), part MOXD1 - monooxygenase, dbh-like 1, KCNK2 - potassium channel, subfamily k, member 2, PLOD2 - procollagen- lysine, 2-oxoglutarate 5-dioxygenase 2, COL23A1 - collagen, type xxiii, alpha 1, UCHL1 - ubiquitin carboxyl-terminal esterase l1 (ubiquitin thiolesterase), COL8A2 - collagen, type viii, alpha 2, COL18A1 - collagen, type xviii, alpha 1, HLA-DPA1 - major histocompatibility complex, class ii, dp alpha 1, PROS1 - protein s (alpha), CYP26B1 - cytochrome p450, family 26, subfamily b, polypeptide 1, ALDH3A2 - aldehyde dehydrogenase 3 family, member a2, EPHX1 - epoxide hydrolase 1, microsomal (xenobiotic), GPX7 - glutathione peroxidase 7, CPT1C - carnitine palmitoyltransferase 1c, RDH5 - retinol dehydrogenase 5 (11-cis/9-cis), MX1 - myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse), PTGIS - prostaglandin i2 (prostacyclin) synthase, COL4A1 - collagen, type iv, alpha 1, HMOX1 - heme oxygenase (decycling) 1, COL11A1 - collagen, type xi, alpha 1, GAS6 - growth arrest-specific 6, PLA2G4C - phospholipase a2, group ivc (cytosolic, calcium-independent), COL15A1 - collagen, type xv, alpha 1] GO:0044421 extracellular 2.65E−13 3.89E−10 2.14 SCPEP1, GSTM2, AKR1C3, PGK1, region part LOXL4, PGM1, GSTM3, COL18A1, PGA5, PGAM1 - phosphoglycerate mutase 1 (brain), ANTXR1 - anthrax toxin receptor 1, TKT - transketolase, SRGN - serglycin, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), CTGF - connective tissue growth factor, DKK3 - dickkopf wnt signaling pathway inhibitor 3, CLDN11 - claudin 11, CCDC80 - coiled-coil domain containing 80, GSTT2 - glutathione s-transferase theta 2, COTL1 - coactosin-like 1 (dictyostelium), ENO2 - enolase 2 (gamma, neuronal), FBLN1 - fibulin 1, CTSK - cathepsin k, AKR1C4 - aldo-keto reductase family 1, member c4, PLOD2 - procollagen-lysine, 2- oxoglutarate 5-dioxygenase 2, CFB - complement factor b, CTSZ - cathepsin z, CCL2 - chemokine (c-c motif) ligand 2, ALDH3A1 - aldehyde dehydrogenase 3 family, member a 1, GREM1 - gremlin 1, dan family bmp antagonist, CLEC3B - c- type lectin domain family 3, member b, ALDH3A2 - aldehyde dehydrogenase 3 family, member a2, VIT - vitrin, APOD - apolipoprotein d, PFKP - phosphofructokinase, platelet, ABI3BP - abi family, member 3 (nesh) binding protein, MYO1D - myosin id, MARCKSL1 - marcks-like 1, F3 - coagulation factor iii (thromboplastin, tissue factor), ALCAM - activated leukocyte cell adhesion molecule, AQP1 - aquaporin 1 (colton blood group), TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b, ADAMTS1 - adam metallopeptidase with thrombospondin type 1 motif, 1, TPI1 - triosephosphate isomerase 1, KRT34 - keratin 34, BAIAP2L1 - bai1-associated protein 2-like 1, EEF1A1 - eukaryotic translation elongation factor 1 alpha 1, ISLR - immunoglobulin superfamily containing leucine-rich repeat, PROS1 - protein s (alpha), WFDC1 - wap four- disulfide core domain 1, SERINC2 - serine incorporator 2, TNFSF4 - tumor necrosis factor (ligand) superfamily, member 4, CXCL16 - chemokine (c-x-c motif) ligand 16, ENPP2 - ectonucleotide pyrophosphatase/phosphodiesterase 2, LUM - lumican, ACYP1 - acylphosphatase 1, erythrocyte (common) type, PTGIS - prostaglandin i2 (prostacyclin) synthase, COL4A1 - collagen, type iv, alpha 1, CD9 - cd9 molecule, TUBB6 - tubulin, beta 6 class v, CYBRD1 - cytochrome b reductase 1, SFRP2 - secreted frizzled-related protein 2, SBSN - suprabasin, UCHL1 - ubiquitin carboxyl-terminal esterase l1 (ubiquitin thiolesterase), QPCT - glutaminyl-peptide cyclotransferase, COL8A2 - collagen, type viii, alpha 2, FAM129A - family with sequence similarity 129, member a, SLIT3 - slit homolog 3 (drosophila), ASS1 - argininosuccinate synthase 1, IGFBP3 - insulin-like growth factor binding protein 3, ADIRF - adipogenesis regulatory factor, BMPER - bmp binding endothelial regulator, MFAP4 - microfibrillar- associated protein 4, THBS4 - thrombospondin 4, PPAP2B - phosphatidic acid phosphatase type 2b, HSPA2 - heat shock 70 kda protein 2, CSRP1 - cysteine and glycine-rich protein 1, HMOX1 - heme oxygenase (decycling) 1, COL11A1 - collagen, type xi, alpha 1, ANPEP - alanyl (membrane) aminopeptidase, GAS6 - growth arrest- specific 6, GSTT2B - glutathione s- transferase theta 2b (gene/pseudogene), FBLN2 - fibulin 2, COL15A1 - collagen, type xv, alpha 1] GO:0070062 extracellular 2.07E−08 6.07E−06 2.04 [SCPEP1 - serine carboxypeptidase 1, vesicular GSTM2 - glutathione s-transferase mu 2 exosome (muscle), AKR1C3 - aldo-keto reductase family 1, member c3, PGK1 - phosphoglycerate kinase 1, LOXL4 - lysyl oxidase-like 4, PGM1 - phosphoglucomutase 1, GSTM3 - glutathione s-transferase mu 3 (brain), COL18A1 - collagen, type xviii, alpha 1, PGA5 - pepsinogen 5, group i (pepsinogen a), PGAM1 - phosphoglycerate mutase 1 (brain), ANTXR1 - anthrax toxin receptor 1, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), TKT - transketolase, CLDN11 - claudin 11, GSTT2 - glutathione s- transferase theta 2, COTL1 - coactosin- like 1 (dictyostelium), ENO2 - enolase 2 (gamma, neuronal), FBLN1 - fibulin 1, AKR1C4 - aldo-keto reductase family 1, member c4, PLOD2 - procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2, CFB - complement factor b, CTSZ - cathepsin z, CLEC3B - c-type lectin domain family 3, member b, ALDH3A2 - aldehyde dehydrogenase 3 family, member a2, APOD - apolipoprotein d, PFKP - phosphofructokinase, platelet, MYO1D - myosin id, F3 - coagulation factor iii (thromboplastin, tissue factor), MARCKSL1 - marcks-like 1, ALCAM - activated leukocyte cell adhesion molecule, AQP1 - aquaporin 1 (colton blood group), KRT34 - keratin 34, TPI1 - triosephosphate isomerase 1, BAIAP2L1 - bai1-associated protein 2-like 1, EEF1A1 - eukaryotic translation elongation factor 1 alpha 1, ISLR - immunoglobulin superfamily containing leucine-rich repeat, PROS1 - protein s (alpha), SERINC2 - serine incorporator 2, LUM - lumican, ACYP1 - acylphosphatase 1, erythrocyte (common) type, CD9 - cd9 molecule, TUBB6 - tubulin, beta 6 class v, CYBRD1 - cytochrome b reductase 1, SBSN - suprabasin, QPCT - glutaminyl- peptide cyclotransferase, FAM129A - family with sequence similarity 129, member a, UCHL1 - ubiquitin carboxyl- terminal esterase l1 (ubiquitin thiolesterase), ASS1 - argininosuccinate synthase 1, IGFBP3 - insulin-like growth factor binding protein 3, MFAP4 - microfibrillar-associated protein 4, ADIRF - adipogenesis regulatory factor, THBS4 - thrombospondin 4, PPAP2B - phosphatidic acid phosphatase type 2b, HSPA2 - heat shock 70 kda protein 2, CSRP1 - cysteine and glycine-rich protein 1, ANPEP - alanyl (membrane) aminopeptidase, GAS6 - growth arrest- specific 6, GSTT2B - glutathione s- transferase theta 2b (gene/pseudogene), FBLN2 - fibulin2, COL15A1 - collagen, type xv, alpha 1] GO:0065010 extracellular 2.07E−08 7.59E−06 2.04 [SCPEP1 - serine carboxypeptidase 1, membrane- GSTM2 - glutathione s-transferase mu 2 bounded (muscle), AKR1C3 - aldo-keto reductase organelle family 1, member c3, PGK1 - phosphoglycerate kinase 1, LOXL4 - lysyl oxidase-like 4, PGM1 - phosphoglucomutase 1, GSTM3 - glutathione s-transferase mu 3 (brain), COL18A1 - collagen, type xviii, alpha 1, PGA5 - pepsinogen 5, group i (pepsinogen a), PGAM1 - phosphoglycerate mutase 1 (brain), ANTXR1 - anthrax toxin receptor 1, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), TKT - transketolase, CLDN11 - claudin 11, GSTT2 - glutathione s- transferase theta 2, COTL1 - coactosin- like 1 (dictyostelium), ENO2 - enolase 2 (gamma, neuronal), FBLN1 - fibulin 1, AKR1C4 - aldo-keto reductase family 1, member c4, PLOD2 - procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2, CFB - complement factor b, CTSZ - cathepsin z, CLEC3B - c-type lectin domain family 3, member b, ALDH3A2 - aldehyde dehydrogenase 3 family, member a2, APOD - apolipoprotein d, PFKP - phosphofructokinase, platelet, MYO1D - myosin id, F3 - coagulation factor iii (thromboplastin, tissue factor), MARCKSL1 - marcks-like 1, ALCAM - activated leukocyte cell adhesion molecule, AQP1 - aquaporin 1 (colton blood group), KRT34 - keratin 34, TPI1 - triosephosphate isomerase 1, BAIAP2L1 - bai1-associated protein 2-like 1, EEF1A1 - eukaryotic translation elongation factor 1 alpha 1, ISLR - immunoglobulin superfamily containing leucine-rich repeat, PROS1 - protein s (alpha), SERINC2 - serine incorporator 2, LUM - lumican, ACYP1 - acylphosphatase 1, erythrocyte (common) type, CD9 - cd9 molecule, TUBB6 - tubulin, beta 6 class v, CYBRD1 - cytochrome b reductase 1, SBSN - suprabasin, QPCT - glutaminyl- peptide cyclotransferase, FAM129A - family with sequence similarity 129, member a, UCHL1 - ubiquitin carboxyl- terminal esterase l1 (ubiquitin thiolesterase), ASS1 - argininosuccinate synthase 1, IGFBP3 - insulin-like growth factor binding protein 3, MFAP4 - microfibrillar-associated protein 4, ADIRF - adipogenesis regulatory factor, THBS4 - thrombospondin 4, PPAP2B - phosphatidic acid phosphatase type 2b, HSPA2 - heat shock 70 kda protein 2, CSRP1 - cysteine and glycine-rich protein 1, ANPEP - alanyl (membrane) aminopeptidase, GAS6 - growth arrest- specific 6, GSTT2B - glutathione s- transferase theta 2b (gene/pseudogene), FBLN2 - fibulin2, COL15A1 - collagen, type xv, alpha 1] GO:0043230 extracellular 2.07E−08 1.01E−05 2.04 [SCPEP1 - serine carboxypeptidase 1, organelle GSTM2 - glutathione s-transferase mu 2 (muscle), AKR1C3 - aldo-keto reductase family 1, member c3, PGK1 - phosphoglycerate kinase 1, LOXL4 - lysyl oxidase-like 4, PGM1 - phosphoglucomutase 1, GSTM3 - glutathione s-transferase mu 3 (brain), COL18A1 - collagen, type xviii, alpha 1, PGA5 - pepsinogen 5, group i (pepsinogen a), PGAM1 - phosphoglycerate mutase 1 (brain), ANTXR1 - anthrax toxin receptor 1, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), TKT - transketolase, CLDN11 - claudin 11, GSTT2 - glutathione s- transferase theta 2, COTL1 - coactosin- like 1 (dictyostelium), ENO2 - enolase 2 (gamma, neuronal), FBLN1 - fibulin 1, AKR1C4 - aldo-keto reductase family 1, member c4, PLOD2 - procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2, CFB - complement factor b, CTSZ - cathepsin z, CLEC3B - c-type lectin domain family 3, member b, ALDH3A2 - aldehyde dehydrogenase 3 family, member a2, APOD - apolipoprotein d, PFKP - phosphofructokinase, platelet, MYO1D - myosin id, F3 - coagulation factor iii (thromboplastin, tissue factor), MARCKSL1 - marcks-like 1, ALCAM - activated leukocyte cell adhesion molecule, AQP1 - aquaporin 1 (colton blood group), KRT34 - keratin 34, TPI1 - triosephosphate isomerase 1, BAIAP2L1 - bai1-associated protein 2-like 1, EEF1A1 - eukaryotic translation elongation factor 1 alpha 1, ISLR - immunoglobulin superfamily containing leucine-rich repeat, PROS1 - protein s (alpha), SERINC2 - serine incorporator 2, LUM - lumican, ACYP1 - acylphosphatase 1, erythrocyte (common) type, CD9 - cd9 molecule, TUBB6 - tubulin, beta 6 class v, CYBRD1 - cytochrome b reductase 1, SBSN - suprabasin, QPCT - glutaminyl- peptide cyclotransferase, FAM129A - family with sequence similarity 129, member a, UCHL1 - ubiquitin carboxyl- terminal esterase l1 (ubiquitin thiolesterase), ASS1 - argininosuccinate synthase 1, IGFBP3 - insulin-like growth factor binding protein 3, MFAP4 - microfibrillar-associated protein 4, ADIRF - adipogenesis regulatory factor, THBS4 - thrombospondin 4, PPAP2B - phosphatidic acid phosphatase type 2b, HSPA2 - heat shock 70 kda protein 2, CSRP1 - cysteine and glycine-rich protein 1, ANPEP - alanyl (membrane) aminopeptidase, GAS6 - growth arrest- specific 6, GSTT2B - glutathione s- transferase theta 2b (gene/pseudogene), FBLN2 - fibulin2, COL15A1 - collagen, type xv, alpha 1] GO:0005576 extracellular 1.74E−04 2.12E−02 2.02 [COL18A1 - collagen, type xviii, alpha 1, region PROS1 - protein s (alpha), SRGN - serglycin, PDGFRL - platelet-derived growth factor receptor-like, CTGF - connective tissue growth factor, OLFML1 - olfactomedin-like 1, CXCL16 - chemokine (c-x-c motif) ligand 16, LUM - lumican, GPX7 - glutathione peroxidase 7, MEGF6 - multiple egf-like-domains 6, COL4A1 - collagen, type iv, alpha 1, PSG6 - pregnancy specific beta-1- glycoprotein 6, FBLN1 - fibulin 1, CTSK - cathepsin k, COL8A2 - collagen, type viii, alpha 2, CFB - complement factor b, PENK - proenkephalin, IL1R1 - interleukin 1 receptor, type i, CCL2 - chemokine (c-c motif) ligand 2, IGFBP3 - insulin-like growth factor binding protein 3, CPA4 - carboxypeptidase a4, MFAP4 - microfibrillar-associated protein 4, THBS4 - thrombospondin 4, CLEC3B - c-type lectin domain family 3, member b, APOD - apolipoprotein d, COL11A1 - collagen, type xi, alpha 1, GAS6 - growth arrest-specific 6, FBLN2 - fibulin 2, COL15A1 - collagen, type xv, alpha 1, TNFRSF11B - tumor necrosis factor receptor superfamily, member 11b] GO:0031982 vesicle 6.79E−08 1.66E−05 1.89 [GSTM2 - glutathione s-transferase mu 2 (muscle), SCPEP1 - serine carboxypeptidase 1, AKR1C3 - aldo-keto reductase family 1, member c3, PGK1 - phosphoglycerate kinase 1, LOXL4 - lysyl oxidase-like 4, GSTM3 - glutathione s-transferase mu 3 (brain), PGM1 - phosphoglucomutase 1, COL18A1 - collagen, type xviii, alpha 1, PGA5 - pepsinogen 5, group i (pepsinogen a), PGAM1 - phosphoglycerate mutase 1 (brain), ANTXR1 - anthrax toxin receptor 1, TKT - transketolase, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), SRGN - serglycin, CLDN11 - claudin 11, GSTT2 - glutathione s-transferase theta 2, COTL1 - coactosin-like 1 (dictyostelium), ENO2 - enolase 2 (gamma, neuronal), FBLN1 - fibulin 1, AKR1C4 - aldo-keto reductase family 1, member c4, PLOD2 - procollagen-lysine, 2-oxoglutarate 5- dioxygenase 2, CFB - complement factor b, CTSZ - cathepsin z, TBC1D2 - tbc1 domain family, member 2, CLEC3B - c- type lectin domain family 3, member b, ALDH3A2 - aldehyde dehydrogenase 3 family, member a2, APOD - apolipoprotein d, PFKP - phosphofructokinase, platelet, MYO1D - myosin id, MARCKSL1 - marcks-like 1, F3 - coagulation factor iii (thromboplastin, tissue factor), ALCAM - activated leukocyte cell adhesion molecule, AQP1 - aquaporin 1 (colton blood group), STMN2 - stathmin-like 2, ADAMTS1 - adam metallopeptidase with thrombospondin type 1 motif, 1, KRT34 - keratin 34, TPI1 - triosephosphate isomerase 1, BAIAP2L1 - bai1-associated protein 2-like 1, EEF1A1 - eukaryotic translation elongation factor 1 alpha 1, ISLR - immunoglobulin superfamily containing leucine-rich repeat, PROS1 - protein s (alpha), SERINC2 - serine incorporator 2, LUM - lumican, ACYP1 - acylphosphatase 1, erythrocyte (common) type, CD9 - cd9 molecule, TRPV2 - transient receptor potential cation channel, subfamily v, member 2, TUBB6 - tubulin, beta 6 class v, AXIN2 - axin 2, CYBRD1 - cytochrome b reductase 1, SBSN - suprabasin, UCHL1 - ubiquitin carboxyl-terminal esterase l1 (ubiquitin thiolesterase), QPCT - glutaminyl-peptide cyclotransferase, FAM129A - family with sequence similarity 129, member a, ASS1 - argininosuccinate synthase 1, IGFBP3 - insulin-like growth factor binding protein 3, ADIRF - adipogenesis regulatory factor, MFAP4 - microfibrillar-associated protein 4, THBS4 - thrombospondin 4, PPAP2B - phosphatidic acid phosphatase type 2b, HSPA2 - heat shock 70 kda protein 2, CSRP1 - cysteine and glycine- rich protein 1, GAS6 - growth arrest- specific 6, ANPEP - alanyl (membrane) aminopeptidase, GSTT2B - glutathione s- transferase theta 2b (gene/pseudogene), FBLN2 - fibulin2, COL15A1 - collagen, type xv, alpha 1] GO:0031988 membrane- 1.18E−07 2.47E−05 1.89 [SCPEP1 - serine carboxypeptidase 1, bounded GSTM2 - glutathione s-transferase mu 2 vesicle (muscle), AKR1C3 - aldo-keto reductase family 1, member c3, PGK1 - phosphoglycerate kinase 1, LOXL4 - lysyl oxidase-like 4, PGM1 - phosphoglucomutase 1, GSTM3 - glutathione s-transferase mu 3 (brain), COL18A1 - collagen, type xviii, alpha 1, PGA5 - pepsinogen 5, group i (pepsinogen a), PGAM1 - phosphoglycerate mutase 1 (brain), ANTXR1 - anthrax toxin receptor 1, CDH2 - cadherin 2, type 1, n-cadherin (neuronal), SRGN - serglycin, TKT - transketolase, CLDN11 - claudin 11, GSTT2 - glutathione s-transferase theta 2, COTL1 - coactosin-like 1 (dictyostelium), ENO2 - enolase 2 (gamma, neuronal), FBLN1 - fibulin 1, AKR1C4 - aldo-keto reductase family 1, member c4, PLOD2 - procollagen-lysine, 2-oxoglutarate 5- dioxygenase 2, CFB - complement factor b, CTSZ - cathepsin z, TBC1D2 - tbc1 domain family, member 2, CLEC3B - c- type lectin domain family 3, member b, ALDH3A2 - aldehyde dehydrogenase 3 family, member a2, APOD - apolipoprotein d, PFKP - phosphofructokinase, platelet, MYO1D - myosin id, F3 - coagulation factor iii (thromboplastin, tissue factor), MARCKSL1 - marcks-like 1, ALCAM - activated leukocyte cell adhesion molecule, AQP1 - aquaporin 1 (colton blood group), KRT34 - keratin 34, TPI1 - triosephosphate isomerase 1, BAIAP2L1 - bai1-associated protein 2-like 1, ISLR - immunoglobulin superfamily containing leucine-rich repeat, EEF1A1 - eukaryotic translation elongation factor 1 alpha 1, PROS1 - protein s (alpha), SERINC2 - serine incorporator 2, LUM - lumican, ACYP1 - acylphosphatase 1, erythrocyte (common) type, TRPV2 - transient receptor potential cation channel, subfamily v, member 2, CD9 - cd9 molecule, TUBB6 - tubulin, beta 6 class v, AXIN2 - axin 2, CYBRD1 - cytochrome b reductase 1, SBSN - suprabasin, FAM129A - family with sequence similarity 129, member a, QPCT - glutaminyl-peptide cyclotransferase, UCHL1 - ubiquitin carboxyl-terminal esterase l1 (ubiquitin thiolesterase), ASS1 - argininosuccinate synthase 1, IGFBP3 - insulin-like growth factor binding protein 3, MFAP4 - microfibrillar-associated protein 4, ADIRF - adipogenesis regulatory factor, THBS4 - thrombospondin 4, PPAP2B - phosphatidic acid phosphatase type 2b, HSPA2 - heat shock 70 kda protein 2, CSRP1 - cysteine and glycine-rich protein 1, ANPEP - alanyl (membrane) aminopeptidase, GAS6 - growth arrest- specific 6, GSTT2B - glutathione s- transferase theta 2b (gene/pseudogene), FBLN2 - fibulin 2, COL15A1 - collagen, type xv, alpha 1]

Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. 

We claim:
 1. A device comprising a hydrogel and a population of fibroblasts, wherein the hydrogel comprises pores, and wherein the population of fibroblasts is seeded into or onto the hydrogel, and wherein the population of fibroblasts comprises diabetic ulcer fibroblast cells.
 2. The device of claim 1, wherein the pores comprise nanopores, micropores, macropores, or a combination thereof.
 3. The device of claim 1, wherein the population of fibroblast is derived from the site of the ulcer or a site adjacent to the ulcer.
 4. The device of claim 1, wherein at least 10% of the fibroblasts in the population are derived from the site of the ulcer or a site adjacent to the ulcer.
 5. The device of claim 4, wherein the population of fibroblasts comprises a fibroblast that is derived from a portion of the skin of the subject.
 6. The device of claim 3, wherein the subject is a mammal.
 7. The device of claim 6, wherein the subject is a human.
 8. The device of claim 1, wherein the population of fibroblasts comprises fibroblasts that have been cultured in vitro.
 9. The device of claim 1, wherein the population of fibroblasts comprises a fibroblast comprising an epigenetic alteration compared to a fibroblast derived from a subject i) not suffering from diabetes, ii) not having a wound, or both i) and ii).
 10. The device of claim 1, wherein the population of fibroblasts comprises a genetically modified fibroblast.
 11. The device of claim 1, further comprising a bioactive composition.
 12. The device of claim 11, wherein the bioactive composition comprises vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), or fibroblast growth factor 2 (FGF2) or a combination thereof.
 13. The device of claim 1, wherein the population of fibroblasts is derived from or isolated from a foot ulcer from a subject suffering from diabetes.
 14. The device of claim 13, wherein the population of fibroblasts expresses fibronectin at a level at least two fold more than nondiabetic, nonulcerated foot-derived fibroblasts.
 15. The device of claim 1, wherein said hydrogel comprises an alginate hydrogel.
 16. A method of treating a wound in a patient in need thereof comprising administering the device of claim
 1. 17. The method of claim 16, wherein the patient suffers from diabetes.
 18. The method of claim 17, wherein the patient suffers from an ulcer.
 19. The method of claim 18, wherein the ulcer is located in an extremity of the patient.
 20. The method of claim 16, wherein the device is administered by injection, implantation, or placement on a wound bed.
 21. The method of claim 16, wherein the population of fibroblasts comprises an autologous fibroblast.
 22. The method of claim 16, wherein the population of fibroblasts comprises an allogeneic or xenogeneic fibroblast.
 23. The method of claim 22, wherein the population of fibroblasts comprises at least 10% autologous fibroblasts.
 24. The method of claim 22, wherein the population of fibroblasts comprises at least 10% allogeneic fibroblasts.
 25. The method of claim 22, wherein the population of fibroblasts comprises at least 10% xenogeneic fibroblasts. 